Foam composite for the repair or regeneration of tissue

ABSTRACT

The present patent describes a biocompatible composite made of a first fibrous layer attached to a three-dimensional inter-connected open cell porous foams that have a gradient in composition and/or microstructure through one or more directions. These composites can be made from blends of absorbable and biocompatible polymers. These biocompatible composites are particularly well suited to tissue engineering applications and can be designed to mimic tissue transition or interface zones.

FIELD OF THE INVENTION

[0001] The present invention relates generally to the field of tissuerepair and regeneration. More particularly the present invention relatesto porous biocompatible bioabsorbable foams that have a gradient incomposition and/or microstructure that serve as a template for tissueregeneration, repair or augmentation.

BACKGROUND OF THE INVENTION

[0002] Open cell porous biocompatible foams have been recognized to havesignificant potential for use in the repair and regeneration of tissue.Early efforts in tissue repair focused on the use of amorphousbiocompatible foam as porous plugs to fill voids in bone. Brekke, et al.(U.S. Pat. No. 4,186,448) described the use of porous mesh plugscomposed of polyhydroxy acid polymers such as polylactide for healingbone voids. Several attempts have been made in the recent past to makeTE scaffolds using different methods, for example U.S. Pat. No.5,522,895 (Mikos) and U.S. Pat. No. 5,514,378 (Mikos, et al.) usingleachables; U.S. Pat. No. 5,755,792 (Brekke) and U.S. Pat. No. 5,133,755(Brekke) using vacuum foaming techniques; U.S. Pat. No. 5,716,413(Walter, et al.) and U.S. Pat. No. 5,607,474 (Athanasiou, et al.) usingprecipitated polymer gel masses; U.S. Pat. No. 5,686,091 (Leong, et al.)and U.S. Pat. No. 5,677,355 (Shalaby, et al.) using polymer melts withfugitive compounds that sublimate at temperatures greater than roomtemperature; and U.S. Pat. No. 5,770,193 (Vacanti, et al.) U.S. Pat. No.5,769,899 (Schwartz, et al.) and U.S. Pat. No. 5,711,960 (Shikinami)using textile-based fibrous scaffolds. Hinsch et al. (EPA 274,898)described a porous open cell foam of polyhydroxy acids with pore sizesfrom about 10 to about 200 μm for the in-growth of blood vessels andcells. The foam described by Hincsh could also be reinforced withfibers, yarns, braids, knitted fabrics, scrims and the like. Hincsh'swork also described the use of a variety of polyhydroxy acid polymersand copolymers such as poly-L-lactide, poly-DL-lactide, polyglycolide,and polydioxanone. The Hincsh foams had the advantage of having regularpore sizes and shapes that could be controlled by the processingconditions, solvents selected, and the additives.

[0003] However, the above techniques have limitations in producing ascaffold with a gradient structure. Most of the scaffolds are isotropicin form and function and lack the anisotropic features of naturaltissues. Further, it is the limitation of prior art to make 3D scaffoldsthat have the ability to control the spatial distribution of variouspore shapes. The process that is described to fabricate themicrostructure controlled foams is a low temperature process that offersmany advantages over other conventional techniques. For example theprocess allows the incorporation of thermally sensitive compounds likeproteins, drugs and other additives with the thermally andhydrolytically unstable absorbable polymers.

[0004] Athanasiou et al. (U.S. Pat. No. 5,607,474) have more recentlyproposed using a two layer foam device for repairing osteochondraldefects at a location where two dissimilar types of tissue are present.The Athanasiou device is composed of a first and second layer, preparedin part separately, and joined together at a subsequent step. Each ofthe scaffold layers is designed to have stiffness and compressibilitycorresponding to the respective cartilage and bone tissue. Sincecartilage and bone often form adjacent layers in the body this approachis an attempt to more clearly mimic the structure of the human body.However, the interface between the cartilage and bone in the human bodyis not a discrete junction of two dissimilar materials with an abruptchange in anatomical features and/or the mechanical properties. Thecartilage cells have distinctly different cell morphology andorientation depending on the location of the cartilage cell in relationto the underlying bone structure. The difference in cartilage cellmorphology and orientation provides a continuous transition from theouter surface of the cartilage to the underlying bone cartilageinterface. Thus the two layer system of Athanasiou, although anincremental improvement, does not mimic the tissue interfaces present inthe human body.

[0005] Another approach to make three-dimensional laminated foams isproposed by Mikos et al. (U.S. Pat. No. 5,514,378). In this techniquewhich is quite cumbersome, a porous membrane is first prepared by dryinga polymer solution containing leachable salt crystals. Athree-dimensional structure is then obtained by laminating severalmembranes together, which are cut to a contour drawing of the desiredshape.

[0006] One of the major weaknesses of the prior art regardingthree-dimensional porous scaffolds used for the regeneration ofbiological tissue like cartilage is that their microstructure is random.These scaffolds, unlike natural tissue, do not vary in morphology orstructure. Further, current scaffolds do not provide adequate nutrientand fluid transport for many applications. Finally, the laminatedstructures are not completely integrated and subjected to delaminationunder in vivo conditions.

[0007] Therefore, it is an object of the present invention to provide abiocompatible, bioabsorbable foam that provides a continuoustransitional gradient of morphological, structural and/or materials.Further, it is preferred that foams used in tissue engineering have astructure that provides organization at the microstructure level thatprovides a template that facilitates cellular invasion, proliferationand differentiation that will ultimately result in regeneration offunctional tissue.

SUMMARY OF INVENTION

[0008] The present invention provides a composite comprising a firstlayer that is a biocompatible filamentous layer and a second layer ofbiocompatible foam. This composite structure allows for the creation ofstructures with unique mechanical properties. The fibrous layer allowsthe composite to have variable mechanical strength depending on thedesign, a different bioabsorption profile, and a differentmicroenvironment for cell invasion and seeding, which are advantageousin a variety of medical applications. The fibrous layer may be made froma variety of biocompatible polymers and blends of biocompatiblepolymers, which are preferably bioabsorbable. The biocompatible foam maybe either a gradient foam or a channeled foam. The gradient foam has asubstantially continuous transition in at least one characteristicselected from the group consisting of composition, stiffness,flexibility, bioabsorption rate, pore architecture and/ormicrostructure. The gradient foam can be made from a blend of absorbablepolymers that form compositional gradient transitions from one polymericmaterial to a second polymeric material. In situations where a singlechemical composition is sufficient for the application, the inventionprovides a composite that may have microstructural variations in thestructure across one or more dimensions that may mimic the anatomicalfeatures of the tissue (e.g. cartilage, skin, bone etc.). The channeledfoam provides channels that extend through the foam to facilitate cellmigration and nutrient flow throughout the channeled foam.

[0009] The present invention also provides a method for the repair orregeneration of tissue contacting a first tissue with the compositedescribed above at a location on the composite that has appropriateproperties to facilitate the growth of said tissue. These compositestructures are particularly useful for the regeneration of tissuebetween two or more different types of tissues. For a multi-cellularsystem in the simplest case, one cell type could be present on one sideof the scaffold and a second cell type on the other side of thescaffold. Examples of such regeneration can be (a) vascular tissue: withsmooth muscle on the outside and endothelial cells on the inside toregenerate vascular structures; (b) meniscal tissue: by implanting withchondrocytes inside foam of the composite and orienting the fibroussurface to the outside.

BRIEF DESCRIPTION OF FIGURES

[0010]FIG. 1 is a scanning electron micrograph of the cross section of arandom microstructure foam made from 5% solution of 35/65ε-caprolactone-co-glycolide copolymer.

[0011]FIG. 2 is a scanning electron micrograph of the cross section of afoam with vertical open channels made from 10% solution of 35/65ε-caprolactone-co-glycolide copolymer.

[0012]FIG. 3 is a scanning electron micrograph of the cross section of afoam with architectural gradient made from 10% solution of 35/65ε-caprolactone-co-glycolide copolymer.

[0013]FIG. 4 is a scanning electron micrograph of the cross section of ag radient foam made from a 50/50 blend of 40/60 ε-caprclactone-co-(L)lactide copolymer and 35/65 ε-caprolactone-co-glycolide copolymer.

[0014]FIG. 5 is a scanning electron micrograph of a cross section of thetop portion of a gradient foam made from a 50/50 blend of 40/60ε-caprolactone-co-(L)lactide copolymer and 35/65ε-caprolactone-co-glycolide copolymer.

[0015]FIG. 6 is a scanning electron micrograph of a cross section of thebottom portion of a gradient foam made from a 50/50 blend of 40/60ε-caprolactone-co-(L)lactide copolymer and 35/65ε-caprolactone-co-glycolide copolymer.

[0016]FIG. 7 is a graphical presentation of cell culture data, 7A, 7Band 7C.

[0017]FIG. 8 is an anatomical sketch of cartilage tissue.

[0018]FIGS. 9A, 9B, and 9C are scanning electron micrographs of a 0.5 mmfoam made from a 50/50 blend of a 35/65 ε-caprolactone-co-glycolidecopolymer and a 40/60 ε-caprolactone-co-(L)lactide copolymer witharchitecture suitable for use as a skin scaffold.

[0019]FIG. 9A shows the porosity of the surface of the scaffold thatpreferably would face the wound bed.

[0020]FIG. 9B shows the porosity of the surface of the scaffolding thatwould preferably face away from the wound bed.

[0021]FIG. 9C shows a cross section of the scaffold with channelsrunning through the thickness of the foam.

[0022]FIG. 10 is a dark field 40× photomicrograph of a trichrome stainedsample illustrating the cellular invasion of the foam shown in FIG. 9,eight days after implantation in a swine model.

[0023]FIG. 11 is a 100× composite photomicrograph of a trichrome stainedsample illustrating the cellular invasion of the foam shown in FIG. 9which also contained PDGF, eight days after implantation in a swinemodel.

[0024]FIG. 12 is a photomicrograph of a cross-section of anelectrostatically spun fibrous layer fused to a foam substrate (bothmaterials are 60/40 copolymers of (L)lactide and ε-caprolactone).

[0025]FIG. 13 is a photomicrograph of the surface porosity of theelectrostatically spun layer shown in FIG. 12.

DETAILED DESCRIPTION OF THE INVENTION

[0026] This invention describes porous bioabsorbable polymer foams thathave novel microstructures. The features of such foams can be controlledto suit a desired application by choosing the appropriate conditions toform the foam during lyophilization. These features in absorbablepolymers have distinct advantages over the prior art where the scaffoldsare typically isotropic or random structures. However, it is preferredthat foams used in tissue engineering (i.e. repair or regeneration) havea structure that provides organization at the microstructural level thatprovides a template that facilitates cellular organization andregeneration of tissue that has the anatomical, biomechanical, andbiochemical features of normal tissues. These foams can be used torepair or regenerate tissue (including organs) in animals such asdomestic animals, primates and humans.

[0027] The features of such foams can be controlled to suit desiredapplication by selecting the appropriate conditions for lyophilizationto obtain one or more of the following properties: (1) interconnectingpores of sizes ranging from about 10 to about 200 μm (or greater) thatprovide pathways for cellular ingrowth and nutrient diffusion; (2) avariety of porosities ranging from about 20% to about 98% and preferablyranging from about 80% to about 95%; (3)gradient in the pore size acrossone direction for preferential cell culturing; (4) channels that runthrough the foam for improved cell invasion, vascularization andnutrient diffusion; (5) micro-patterning of pores on the surface forcellular organization; (6) tailorability of pore shape and/ororientation (e.g. substantially spherical, ellipsoidal, columnar); (7)anisotropic mechanical properties; (8) composite foams with a polymercomposition gradient to elicit or take advantage of different cellresponse to different materials; (9) blends of different polymercompositions to create structures that have portions that will breakdown at different rates; (10) foams co-lyophilized or coated withpharmaceutically active compounds including but not limited tobiological factors such as RGD's, growth factors (PDGF, TGF-β, VEGF,BMP, FGF etc.) and the like; (11) ability to make 3 dimensional shapesand devices with preferred microstructures; and (12) lyophilization withother parts or medical devices to provide a composite structure. Thesecontrolled features in absorbable polymers have distinct advantages overthe prior art where the scaffolds are typically isotropic or randomstructures with no preferred morphology at the pore level. However, itis preferred that foams used in tissue scaffolds have a structure thatprovides organization at the microstructure level and provides atemplate that facilitates cellular organization that may mimic naturaltissue. The cells will adhere, proliferate and differentiate along andthrough the contours of the structure. This will ultimately result in acultured tissue that may mimic the anatomical features of real tissuesto a large extent.

[0028] For example, as shown in FIG. 3 the orientation of the major axisof the pores may be changed from being in the same plane as the foam tobeing oriented perpendicular to the plane of the foam. As can be seenfrom FIG. 3 the pore size can be varied from a small pore size generallybetween about 30 μm and about 50 μm to a larger size of from about 100μm to about 200 μm in porous gradient foams. Ideally the foam structurecould be created to facilitate the repair or regeneration of humantissue junctions such as the cartilage to bone junction present injoints. This foam would progress from a small (i.e. about 30 μm to about150 μm in diameter) round pores to larger column-like pores (i.e. about30 μm to about 400 μm in diameter, preferably about 100 μm to about 400μm in diameter, in most cases with a length to diameter ratio of atleast 2). Foams with channels are illustrated in FIG. 2 and FIG. 3.

[0029] The channels formed by this process generally begin on onesurface of the foam and may traverse the thickness of the foam. Thechannel's length is generally at least two times the average porediameter and preferably are at least four times the average porediameter and most preferably at least eight times the average porediameter. Channels for most applications will be at least 200 microns inlength and may extend through the thickness of the foam. The diameter ofthe channel will be at least one time the size of the average porediameter and preferably at least 2 to 3 times the average pore diameter.The channel size and diameter of course will be selected based on thedesired functionality of the channel such as cellular invasion, nutrientdiffusion or as an avenue for vascularization.

[0030] There are a number of biological tissues that demonstrategradient architectures. Examples of tissues where a gradient scaffoldcould be used, include, but are not limited to: bone, spine disc,articular cartilage, meniscus, fibrocartilage, tendons, ligaments, dura,skin, vascular grafts, nerves, liver, and pancreas. The examples belowonly highlight a few tissues where gradient scaffolds could be used. Thedesign of tissue engineered scaffolds to facilitate development of theseorgan structures would benefit greatly from the ability to process orcreate a gradient architecture in the scaffold.

[0031] Cartilage

[0032] Articular cartilage covers the ends of all bones that formarticulating joints in humans and animals. The cartilage acts in thejoint as a mechanism for force distribution and as a bearing surfacebetween different bones. Without articular cartilage, stressconcentration and friction would occur to the degree that the jointwould not permit ease of motion. Loss of the articular cartilage usuallyleads to painful arthritis and decreased joint motion. A schematicshowing the morphological features of a healthy cartilage is shown inFIG. 8.

[0033] Articular cartilage is an excellent example of a naturallyoccurring gradient structure. Articular cartilage is composed of fourdifferent zones that include the superficial or tangential zone withinthe first 10-20% of the structure (this includes the articular surface),the middle zone which is 40-60% of the middle structure, and the deepzone that is adjacent to the tide mark, and a transition zone betweenthe bone and cartilage that is composed of calcified cartilage.Subchondral bone is located adjacent to the tide mark and thistransitions into cancellous bone. In the superficial or tangential zone,the collagen fibrils are parallel to the surface. The fibers areoriented to resist shear forces generated during normal jointarticulation. The middle zone has a randomly arranged organization ofmuch larger diameter collagen fibers. Finally, in the deep zone thereare larger collagen fiber bundles, which are perpendicular to thesurface, and they insert into the calcified cartilage. The cells aresperoidiol and tend to arrange themselves in a columnar manner. Thecalcified cartilage zone has smaller cells with relatively littlecytoplasm.

[0034] A preferred embodiment of this invention would be to generate agradient foam structure that could act as a template for multipledistinct zones. These foam structures could be fabricated in a varietyof shapes to regenerate or repair osteochondrial defects and cartilage.One potential foam structure would be cylindrical in shape with anapproximate dimensions of 10 mm in diameter and 10 mm in depth. The topsurface is would be approximately 1 mm thick and would be a low porositylayer to control the fluid permeability. By adopting a suitableprocessing method the surface porosity of the foam could be controlled.The porosity of this skin like surface can be varied from completelyimpervious to completely porous. Fluid permeability would be controlledby surface porosity. Below such a skin the structure would consist ofthree zones. An upper porous zone which lies adjacent to cartilagetissue, a lower porous zone which lies adjacent to bone tissue, and atransition zone between the upper and lower porous zones. For articularcartilage, it is currently preferred that the stiffness (modulus) of theupper and lower porous layers at the time of implantation be at least asstiff, as the corresponding adjacent tissue. In such a case the porouslayers will be able to support the environmental loading and therebyprotect the invading cells until they have differentiated andconsolidated into tissue that is capable of sustaining load. For examplethe porous structure used for the superficial tangential zone could haveelongated pores and the orientation of the structure could be parallelto the surface of the host cartilage. However, the deep zone may have aporosity of about 80 to about 95% with pores that are of the order of100 μm (about 80 μm to about 120 μm). It is expected that chondrocyteswill invade this zone. Below this, would be a zone with larger pores(about 100 μm to about 200 μm) and a porosity in the range of about 50to about 80%. Such 100 μm to about 200 μm porous foam would have astructure such that the struts or walls of the pores are larger andvertical to the load, similar to the naturally occurring structure andto bear the loads. Finally, at the bottom of this structure there is aneed for larger pores (about 150 μm to about 300 μm) with higherstiffness to be structurally compatible with cancellous bone. The foamin this section could be reinforced with ceramic particles or fibersmade up of calcium phosphates and the like.

[0035] Recent data generated in our laboratories support the hypothesisthat cell invasion can be controlled by pore size. In these studies, ascaffold made of 95/5 mole percent poly(L)lactide-co-ε-caprolactone)with an approximate pore size of about 80 μm had chondrocyte invasion ofabout 30 cells/mm² of the scaffold (under static conditions). Scaffoldsmade of 40/60 mole percent poly(ε-caprolactone-co-(L)lactide) with alarger approximate pore size of about 100 μm had a statisticallysignificantly greater cellular invasion of 50 cells/mm² (under staticconditions). In both cases the cells were bovine chondrocytes. A verysimple gradient structure with a variation of pore sized from about 80μm to about 150 μm would provide a structure where chondrocytes wouldmore easily invade the area with larger pores. The area with smallerpores would be void of chondrocytes or would be filled with a secondcell types (e.g., fibroblasts).

[0036] In a compositionally gradient foam a blend of two or moreelastomeric copolymers or in combination with high modulussemi-crystalline polymers along with additives such as growth factors orparticulates can be chosen such that first a desired pore gradient isdeveloped with a preferred spatial organization of the additives. Thenusing a variety of the approaches referred to in the preferred methodsof making gradient foams, a compositional gradient can be superimposedprimarily due to the differences in the polymer-solvent phase separationbehavior of each system. Such a gradient foam structure would elicit afavorable response to chondrocytes or osteoblasts depending on thespatial location.

[0037] Further, the purpose of a functional gradient is to more evenlydistribute the stresses across a region through which mechanical and/orphysical properties are varying and thereby alleviate the stressconcentrating effects of a sudden interface. This more closely resemblesthe actual biological tissues and structures, where structuraltransitions between differing tissues such as cartilage and bone aregradual. Therefore, it is an object of the present invention to providean implant with a functional gradient between material phases. Thepresent invention provides a multi-phasic functionally gradedbioabsorbable implant with attachment means for use in surgical repairof osteochondral defects or sites of osteoarthritis. Several patentshave proposed systems for repairing cartilage that could be used withthe present inventive porous scaffolds. For example, U.S. Pat. No.5,769,899 describes a device for repairing cartilage defects and U.S.Pat. No. 5,713,374 describes securing cartilage repair devices with boneanchors (both hereby incorporated herein by reference).

[0038] Bone

[0039] Gradient structures naturally occur for the bone/cartilageinterface. In a study in our laboratories, we have demonstrated thatmaterial differences significantly influence cell function. In initialand long-term response of primary osteoblasts to polymer films (95/5L-lactide-co-glycolide copolymer, 90/10 glycolide-co-(L)lactidecopolymer, 95/5 L-lactide-co-ε-caprolactone copolymer, 75/25glycolide-co-(L)lactide copolymer and 40/60 ε-caprolactone-co-(L)lactide copolymer and knitted meshes (95/5 (L)lactide-co-glycolide and90/10 glycolide-co-(L)lactide copolymers) were evaluated in vitro. Theresults demonstrated that osteoblasts attached and proliferated well onall the biodegradable polymer films and meshes following 6-dayincubation. None of the tested polymer films, except a 40/60ε-caprolactone-co-(L)lactide copolymer film, demonstrated significantenhancement in differentiation of primary rat osteoblasts as compared totissue culture polystyrene (control). Films made of40/60-caprolactone-co-(L)lactide promoted enhanced differentiation ofcultured osteoblasts as demonstrated by increased alkaline phosphataseactivity and osteoclacin mRNA expression as compared to the other filmsand TCPS. Hence, it is clear that different absorbable materials willsignificantly alter cell function and differentiation. By identifyingthe optimal materials for cell growth and differentiation a compositematerials with a gradient composition could be utilized to optimizetissue regeneration with different cell types in the same scaffold.

[0040] Therefore, for bone repair or regeneration devices orscaffoldings, a device made from a homopolymer, copolymer (random,block, segmented block, tappered blocks, graft, triblock, etc.) having alinear, branched or star structure containing ε-caprolactone isespecially preferred. Currently preferred are aliphatic polyestercopolymers containing in the range of from about 30 weight percent toabout 99 weight percent ε-caprolactone. Suitable repeating units thatmay be copolymerized with ε-caprolactone are well known in the art.Suitable comonomers that may be copolymerized with ε-caprolactoneinclude, but are not limited to lactic acid, lactide (including L-, D-,meso and D,L mixtures), glycolic acid, glycolide, p-dioxanone(1,4-dioxan-2-one), trimethylene carbonate (1,3-dioxan-2-one),δ-valerolactone, β-butyrolactone, ε-decalactone, 2,5-diketomorpholine,pivalolactone, α,α-diethylpropiolactone, ethylene carbonate, ethyleneoxalate, 3-methyl-1,4-dioxane-2,5-dione,3,3-diethyl-1,4-dioxan-2,5-dione, 7-butyrolactone, 1,4-dioxepan-2-one,1,5-dioxepan-2-one, 6,6-dimethyl-dioxepan-2-one,6,8-dioxabicycloctane-7-one and combinations thereof.

[0041] Preferred medical devices or tissue scaffoldings for bone tissuerepair and/or regeneration containing bioabsorbable polymers made fromε-caprolactone include but are not limited to the porous foamscaffoldings (such as described in this application), fibrous threedimensional, spun, nonwoven, woven, knitted, or braided tissuescaffoldings, composite containing reinforcing fibers, matrices andcombinations thereof.

[0042] Skin

[0043] Another example of a tissue that has a gradient structure isskin. The basic structure of skin has two distinct, but well integratedlayers where the thickness of each layer varies at different locationsof the body. The outer layer or epidermis, is avascular and mainlyconsists of keratinocytes with smaller numbers of immune cells(Langerhan cells) and pigmented cells (melanocytes). The keratinocytesproduce keratin fibers and corneocyte envelopes, which gives theepidermis its durability and protective capabilities. The development ofthese structures is completely dependent upon the differentiation stateof the epidermis. The epidermis forms a stratified epithelium, withdifferent protein expression patterns, as the cells move further awayfrom the basement membrane. This stratified layer of differentiallyexpressing cells must be formed for maintenance of epidermal function.Below the epidermis is the dermis, which is a dense irregular connectivetissue that is highly vascular. This layer is heavily populated withcollageneic and elastic fibers, which give it its exceptional elasticityand strength. Fibroblasts are the main cell types in this layer. Betweenthese two layers is the basement membrane, which serves as the site ofattachment for epidermal cells and serves also to regulate theirfunction and differentiation. The layer of keratinocytes, which attachesdirectly to the basement membrane, are cuboidal in shape and highlyaligned. This attachment and architecture are critical requirementsdriving the ultimate production of the higher squamous structures in theepidermis. The basal layer provides a source of precursor cells forrepair and replacement of the epidermis. The squamous layers providestrength and resistance to insult and infection.

[0044] Any material used for replacement of skin must be able to enticeinvasion of fibroblasts or other cells necessary to produce the dermalcomponents of the healed tissue. Additionally, the material must notinhibit, and preferably should enhance, the rate of re-epithelializationin such a fashion that a discreet, epidermal basal layer is formed.Materials that permit invasion into the scaffold by migratingkeratinocytes can produce partially differentiated cells. Consequently,control of access of particular cell types and a porous design thatfacilitates the regeneration of the natural tissue can have functionalbenefits. Now refer to FIGS. 9A, 9B and 9C which illustrates themicrostructure of this foam scaffold. FIGS. 10 (10× magnification) and11 (40× magnification composite picture) provide photomicrographicevidence of the invasion of fibroblasts, macrophages, macrophage giantcells and endothelial-like cells into the a 0.5 mm foam. The foam tissuescaffolding 101 shown in both pictures was a 50:50 blend ofε-caprolactone-co-glycolide copolymer and ε-caprolactone-co-lactidecopolymer (made as described in Example 7). The pictures were taken at 8days after implantation in 1.5 cm×1.5 cm×0.2 cm excisional wound modelin a Yorkshire pig model. Complete incorporation of the matrix into thegranulation tissue bed is evident in both pictures. The dense fibroustissue above the foam tissue scaffolding appears to provide a suitablesubstrate for the over growth of epidermis. PDGF was incorporated intothe foam tissue scaffolding shown in FIG. 11. In compromised woundhealing models the addition of a growth factor such as PDGF may in factbe necessary.

[0045] From our initial studies it appears that it is desirable to useas a skin scaffold a foam tissue scaffold having a thickness of fromabout 150 μm to about 3 mm, preferably the thickness of the foam may bein the range of from about 300 μm to about 1500 μm and most preferablyabout 500 to about 1000 μm. Clearly different skin injuries (i.e.diabetic ulcers, venous stasis ulcers, decubitis ulcers, burns etc.) mayrequire different foam thickness. Additionally, the patient's conditionmay necessitate the incorporation of growth factors, antibiotics andantifungal compounds to facilitate wound healing.

[0046] Vascular Grafts

[0047] The creation of tubular structures with gradients may also be ofinterest. In vascular grafts, having a tube with pores in the outerdiameter which transitions to smaller pores on the inner surface or visaversa may be useful in the culturing of endothelial cells and smoothmuscle cells for the tissue culturing of vessels.

[0048] Multilayered tubular structures allow the regeneration of tissuethat mimics the mechanical and/or biological characteristics of bloodvessels will have utility as a vascular grafts. Concentric layers, madefrom different compositions under different processing conditions couldhave tailored mechanical properties, bioabsorption properties, andtissue ingrowth rates. The inner most, or luminal layer would beoptimized for endothelialization through control of the porosity of thesurface and the possible addition of a surface treatment. The outermost,or adventitial layer of the vascular graft would be tailored to inducetissue ingrowth, again by optimizing the porosity (percent porosity,pore size, pore shape and pore size distribution) and by incorporatingbioactive factors, pharmaceutical agents, or cells. There may or may notbe a barrier layer with low porosity between these two porous layers toincrease strength and decrease leakage.

[0049] Composition of Foams

[0050] A variety of absorbable polymers can be used to make foams.Examples of suitable biocompatible, bioabsorbable polymers that could beused include polymers selected from the group consisting of aliphaticpolyesters, poly(amino acids), copoly(ether-esters), polyalkylenesoxalates, polyamides, poly(iminocarbonates), polyorthoesters,polyoxaesters, polyamidoesters, polyoxaesters containing amine groups,poly(anhydrides), polyphosphazenes, biomolecules (i.e. biopolymers suchaas collagen, elastin, bioabsorbable starches, ect.) and blends thereof.For the purpose of this invention aliphatic polyesters include but arenot limited to homopolymers and copolymers of lactide (which includeslactic acid, D-,L- and meso lactide), glycolide (including glycolicacid), ε-caprolactone, p-dioxanone (1,4-dioxan-2-one), trimethylenecarbonate (1,3-dioxan-2-one), alkyl derivatives of trimethylenecarbonate, δ-valerolactone,β-butyrolactone, γ-butyrolactone,ε-decalactone, hydroxybutyrate (repeating units), hydroxyvalerate(repeating units), 1,4-dioxepan-2-one (including its dimer1,5,8,12-tetraoxacyclotetradecane-7,14-dione), 1,5-dioxepan-2-one,6,6-dimethyl-1,4-dioxan-2-one 2,5-diketomorpholine, pivalolactone,alpha, alpha-diethylpropiolactone, ethylene carbonate, ethylene oxalate,3-methyl-1,4-dioxane-2,5-dione, 3,3-diethyl-1,4-dioxan-2,5-dione,6,8-dioxabicycloctane-7-one and polymer blends thereof.Poly(iminocarbonate) for the purpose of this invention include asdescribed by Kemnitzer and Kohn, in the Handbook of BiodegradablePolymers, edited by Domb, Kost and Wisemen, Hardwood Academic Press,1997, pages 251-272. Copoly(ether-esters) for the purpose of thisinvention include those copolyester-ethers described in “Journal ofBiomaterials Research”, Vol. 22, pages 993-1009, 1988 by Cohn and Younesand Cohn, Polymer Preprints (ACS Division of Polymer Chemistry) Vol.30(1), page 498, 1989 (e.g. PEO/PLA). Polyalkylene oxalates for thepurpose of this invention include U.S. Pat. Nos. 4,208,511; 4,141,087;4,130,639; 4,140,678; 4,105,034; and 4,205,399 (incorporated byreference herein). Polyphosphazenes, co-, ter- and higher order mixedmonomer based polymers made from L-lactide, D,L-lactide, lactic acid,glycolide, glycolic acid, para-dioxanone, trimethylene carbonate andε-caprolactone such as are described by Allcock in The Encyclopedia ofPolymer Science, Vol. 13, pages 31-41, Wiley Intersciences, John Wiley &Sons, 1988 and by Vandorpe, Schacht, Dejardin and Lemmouchi in theHandbook of Biodegradable Polymers, edited by Domb, Kost and Wisemen,Hardwood Academic Press, 1997, pages 161-182 (which are herebyincorporated by reference herein). Polyanhydrides from diacids of theform HOOC—C₆H₄—O—(CH₂)_(m)—O—C₆H₄—COOH where m is an integer in therange of from 2 to 8 and copolymers thereof with aliphatic alpha-omegadiacids of up to 12 carbons.

[0051] Polyoxaesters, polyoxaamides and polyoxaesters containing aminesand/or amido groups are described in one or more of the following U.S.Pat. Nos. 5,464,929; 5,595,751; 5,597,579; 5,607,687; 5,618,552;5,620,698; 5,645,850; 5,648,088; 5,698,213; 5,700,583; and 5,859,150(which are incorporated herein by reference). Polyorthoesters such asthose described by Heller in Handbook of Biodegradable Polymers, editedby Domb, Kost and Wisemen, Hardwood Academic Press, 1997, pages 99-118(hereby incorporated herein by reference).

[0052] Currently aliphatic polyesters are the absorbable polymers thatare preferred for making gradient foams. Aliphatic polyesters can behomopolymers, copolymers (random, block, segmented, tappered blocks,graft, triblock,etc.) having a linear, branched or star structure.Preferred are linear copolymers. Suitable monomers for making aliphatichomopolymers and copolymers may be selected from the group consistingof, but are not limited, to lactic acid, lactide (including L-, D-, mesoand D,L mixtures), glycolic acid, glycolide, ε-caprolactone, p-dioxanone(1,4-dioxan-2-one), trimethylene carbonate (1,3-dioxan-2-one),delta-valerolactone, beta-butyrolactone, epsilon-decalactone,2,5-diketomorpholine, pivalolactone, alpha, alpha-diethylpropiolactone,ethylene carbonate, ethylene oxalate, 3-methyl-1,4-dioxane-2,5-dione,3,3-diethyl-1,4-dioxan-2,5-dione, gamma-butyrolactone,1,4-dioxepan-2-one, 1,5-dioxepan-2-one, 6,6-dimethyl-dioxepan-2-one,6,8-dioxabicycloctane-7-one and combinations thereof.

[0053] Elastomeric copolymers also are particularly useful in thepresent invention. Suitable bioabsorbable biocompatible elastomersinclude but are not limited to those selected from the group consistingof elastomeric copolymers of E-caprolactone and glycolide (preferablyhaving a mole ratio of ε-caprolactone to glycolide of from about 35:65to about 65:35, more preferably from 45:55 to 35:65) elastomericcopolymers of ε-caprolactone and lactide, including L-lactide, D-lactideblends thereof or lactic acid copolymers (preferably having a mole ratioof ε-caprolactone to lactide of from about 35:65 to about 65:35 and morepreferably from 45:55 to 30:70 or from about 95:5 to about 85:15)elastomeric copolymers of p-dioxanone (1,4-dioxan-2-one) and lactideincluding L-lactide, D-lactide and lactic acid (preferably having a moleratio of p-dioxanone to lactide of from about 40:60 to about 60:40)elastomeric copolymers of ε-caprolactone and p-dioxanone (preferablyhaving a mole ratio of ε-caprolactone to p-dioxanone of from about from30:70 to about 70:30) elastomeric copolymers of p-dioxanone andtrimethylene carbonate (preferably having a mole ratio of p-dioxanone totrimethylene carbonate of from about 30:70 to about 70:30), elastomericcopolymers of trimethylene carbonate and glycolide (preferably having amole ratio of trimethylene carbonate to glycolide of from about 30:70 toabout 70:30), elastomeric copolymer of trimethylene carbonate andlactide including L-lactide, D-lactide, blends thereof or lactic acidcopolymers (preferably having a mole ratio of trimethylene carbonate tolactide of from about 30:70 to about 70:30) and blends thereof. Examplesof suitable bioabsorbable elastomers are described in U.S. Pat. Nos.4,045,418; 4,057,537 and 5,468,253 all hereby incorporated by reference.These elastomeric polymers will have an inherent viscosity of from about1.2 dL/g to about 4 dL/g, preferably an inherent viscosity of from about1.2 dL/g to about 2 dL/g and most preferably an inherent viscosity offrom about 1.4 dL/g to about 2 dL/g as determined at 25° C. in a 0.1gram per deciliter (g/dL) solution of polymer in hexafluoroisopropanol(HFIP).

[0054] Preferably, the elastomers will exhibit a high percent elongationand a low modulus, while possessing good tensile strength and goodrecovery characteristics. In the preferred embodiments of thisinvention, the elastomer from which the foams are formed will exhibit apercent elongation greater than about 200 percent and preferably greaterthan about 500 percent. There properties, which measure the degree ofelasticity of the bioabsorbable elastomer, are achieved whilemaintaining a tensile strength greater than about 500 psi, preferablygreater than about 1,000 psi, and a tear strength of greater than about50 lbs/inch, preferably greater than about 80 lbs/inch.

[0055] The polymer or copolymer suitable for forming a gradient foam fortissue regeneration depends on several factors. The chemicalcomposition, spatial distribution of the constituents, the molecularweight of the polymer and the degree of crystallinity all dictate tosome extent the in-vitro and in-vivo behavior of the polymer. However,the selection of the polymers to make gradient foams for tissueregeneration largely depends on (but not limited to) the followingfactors: (a) bio-absorption (or biodegradation) kinetics; (b) in-vivomechanical performance; and (c) cell response to the material in termsof cell attachment, proliferation, migration and differentiation and (d)biocompatibility.

[0056] The ability of the material substrate to resorb in a timelyfashion in the body environment is critical. But the differences in theabsorption time under in-vivo conditions can also be the basis forcombining two different copolymers. For example a copolymer of 35:65ε-caprolactone and glycolide (a relatively fast absorbing polymer) isblended with 40:60 ε-caprolactone and (L)lactide copolymer (a relativelyslow absorbing polymer) to form a foam. Such a foam could have severaldifferent physical structures depending upon the processing techniqueused. The two constituents can be either randomly inter-connectedbicontinuous phases, or the constituents can have a gradient through thethickness or a laminate type composite with a well integrated interfacebetween the two constituent layers. The microstructure of these foamscan be optimized to regenerate or repair the desired anatomical featuresof the tissue that is being engineered.

[0057] One preferred embodiment of the present invention is to usepolymer blends to form structures which transition from one compositionto another composition in a gradient like architecture. Foams havingthis gradient architecture are particularly advantageous in tissueengineering applications to repair or regenerate the structure ofnaturally occurring tissue such as cartilage (articular, meniscal,septal, tracheal etc.), esophaguses, skin, bone and vascular tissue. Forexample by blending an elastomer of ε-caprolactone-co-glycolide withε-caprolactone-co-lactide (i.e. with a mole ratio of about 5:95) a foammay be formed that transitions from a softer spongy foam to a stiffermore rigid foam similar to the transition from cartilage to bone.Clearly other polymer blends may be used for similar gradient effects orto provide different gradients such as different absorption profiles,stress response profiles, or different degrees of elasticity.Additionally, these foams can be used for organ repair replacement orregeneration strategies that may benefit from these unique scaffolds,including but are not limited to, spine disc, dura, nerve tissue, liver,pancreas, kidney, bladder, tendons, ligaments and breast tissues.

[0058] These elastomeric polymers may be foamed by lyophilization,supercritical solvent foaming (i.e., as described in EP 464,163 B1), gasinjection extrusion, gas injection molding or casting with anextractable material (i.e., salts, sugar or any other means known tothose skilled in the art). Currently it is preferred to preparebioabsorbable, biocompatible elastomeric foams by lyophilization.Suitable methods for lyophilizing elastomeric polymers to form foams isdescribed in the Examples and in the copending patent applicationentitled, “Process for Manufacturing Biomedical Foams”, assigned toEthicon, Inc., docket number ETH-1352, filed Jun. 30, 1999 herebyincorporated herein by reference herein.

[0059] The foams that are made in this invention are made by apolymer-solvent phase separation technique with modifications to theprior art that unexpectedly creates gradients in the foam structure.Generally, a polymer solution can be separated into two phases by anyone of the four techniques: (a) thermally inducedgelation/crystalization; (b) non-solvent induced separation of solventand polymer phases; (c) chemically induced phase separation, and (d)thermally induced spinodal decomposition. The polymer solution isseparated in a controlled manner into either two distinct phases or twobicontinuous phases. Subsequent removal of the solvent phase usuallyleaves a porous structure of density less than the bulk polymer andpores in the micrometer ranges (ref. “Microcellular foams via phaseseparation” by A. T. Young, J. Vac. Sci. Technolol. A 4(3), May/Jun1986). The steps involved in the preparation of these foams consists ofchoosing the right solvents for the polymers that needs to belyophilized and preparing a homogeneous solution. Next, the polymersolution is subjected to a freezing and vacuum drying cycle. Thefreezing step phase separates the polymer solution and vacuum dryingstep removes the solvent by sublimation and/or drying leaving a porouspolymer structure or an interconnected open cell porous foam.

[0060] Suitable solvents that should be generally suited as a startingplace for selecting a solvent for the preferred absorbable aliphaticpolyesters include but are not limited to solvents selected from a groupconsisting of formic acid, ethyl formate, acetic acid,hexafluoroisopropanol (HFIP),cyclic ethers (i.e. THF, DMF, and PDO),acetone, acetates of C2 to C5 alcohol (such as ethyl acetate andt-butylacetate),glyme (i.e. monoglyme, ethyl glyme, diglyme, ethyldiglyme, triglyme, butyl diglyme and tetraglyme) methylethyl ketone,dipropyleneglycol methyl ether, lactones (such as γ-valerolactone,δ-valerolactone, β-butyrolactone, γ-butyrolactone)1,4-dioxane,1,3-dioxolane, 1,3-dioxolane-2-one (ethylene carbonate),dimethlycarbonate, benzene, toluene, benzyl alcohol, p-xylene,naphthalene, tetrahydrofuran, N-methyl pyrrolidone, dimethylformamide,chloroform, 1,2-dichloromethane, morpholine, dimethylsulfoxide,hexafluoroacetone sesquihydrate (HFAS), anisole and mixtures thereof.Among these solvents, the preferred solvent is 1,4-dioxane. Ahomogeneous solution of the polymer in the solvent is prepared usingstandard techniques.

[0061] Accordingly, as will be appreciated, the applicable polymerconcentration or amount of solvent, which may be utilized, will varywith each system. Suitable phase diagram curves for several systems havealready been developed. However, if an appropriate curve is notavailable, this can be readily developed by known techniques. Forexample, a suitable technique is set forth in Smolders, van Aartsen andSteenbergen, Kolloid-Z. u. Z. Polymere, 243, 14 (1971). As a generalguideline the amount of polymer in the solution can vary from about 0.5%to about 90% and preferably will vary from about 0.5% to about 30% byweight depending to a large extent on the solubility of the polymer in agiven solvent and the final properties of the foam desired.

[0062] Additionally, solids may be added to the polymer-solvent system.The solids added to the polymer-solvent system preferably will not reactwith the polymer or the solvent. Suitable solids include materials thatpromote tissue regeneration or regrowth, buffers, reinforcing materialsor porosity modifiers. Suitable solids include, but are not limited to,particles of demineralized bone, calcium phosphate particles, or calciumcarbonate particles for bone repair, leachable solids for pore creationand particles of bioabsorbable polymers not soluble in the solventsystem as reinforcing or to create pores as they are absorbed. Suitableleachable solids include but are not limited nontoxic leachablematerials selected from the group consisting of salts (i.e. sodiumchloride, potassium chloride, calcium chloride, sodium tartrate, sodiumcitrate, and the like) biocompatible mono and disaccharides(i.e.glucose, fructose, dextrose, maltose, lactose and sucrose),polysaccharides (i.e. starch, alginate), water soluble proteins(i.e.gelatin and agarose). Generally all of these materials will have anaverage diameter of less than about 1 mm and preferably will have anaverage diameter of from about 50 to about 500 μm. The particles willgenerally constitute from about 1 to about 50 volume percent of thetotal volume of the particle and polymer-solvent mixture (wherein thetotal volume percent equals 100 volume percent). The leachable materialscan be removed by immersing the foam with the leachable material in asolvent in which the particle is soluble for a sufficient amount of timeto allow leaching of substantially all of the particles, but which doesnot dissolve or detrimentally alter the foam. The preferred extractionsolvent is water, most preferably distilled-deionized water. Thisprocess is described in U.S. Pat. No. 5,514,378 hereby incorporatedherein by reference (see column 6). Preferably the foam will be driedafter the leaching process is complete at low temperature and/or vacuumto minimize hydrolysis of the foam unless accelerated absorption of thefoam is desired.

[0063] After the polymer solvent mixture is formed the mixture is thensolidified. For a specific polymer-solvent system, the solidificationpoint, the melt temperature and the apparent glass transition of thepolymer-solvent system can be determined using standard differentialscanning calorimetric (DSC) techniques. In theory, but in no waylimiting the scope of the present invention, it is believed that as apolymer solvent system is cooled down an initial solidification occursat about or below the freezing point of the solvent. This corresponds tothe freezing of a substantial portion of the solvent in the system. Theinitial freezing appears as a first exothermic peak. A second freezingpoint occurs when the remaining solvent associated with the polymersolidifies. The second freezing point is marked by a second exothermicpeak. The apparent Tg is the temperature at which the fully frozensystem displays the first endothermic shift on reheating.

[0064] An important parameter to control is the rate of freezing of thepolymer-solvent system. The type of pore morphology that gets locked induring the freezing step is a function of the solution thermodynamics,freezing rate, temperature to which it is cooled, concentration of thesolution, homogeneous or heterogenous nucleation etc. Detaileddescription of these phase separation phenomenon can be found in thereferences provided herein (“Microcellular foams via phase separation”by A. T. Young, J. Vac. Sci. Technol. A 4(3), May/June 1986; and“Thermodynamics of Formation of Porous Poymeric Membrane from Solutions”by S. Matsuda, Polymer J. Vol. 23, No. 5, pp 435-444, 1991).

[0065] The polymer solution previously described can be solidified in avariety of manners such as placing or injecting the solution in a moldand cooling the mold in an appropriate bath or on a refrigerated shelf.Alternatively, the polymer solution can be atomized by an atomizer andsprayed onto a cold surface causing solidification of the spray layer bylayer. The cold surface can be a medical device or part thereof or afilm. The shape of the solidified spray will be similar to the shape ofthe surface it is sprayed onto. Alternatively, the mixture aftersolidification can be cut or formed to shape while frozen. Using theseand other processes the foams can be made or molded in a variety ofshapes and sizes (i.e. tubular shapes, branched tubular shapes,spherical shapes, hemispherical shapes, three-dimensional polygonalshapes, ellipsoidal shapes (i.e. kidney shaped, toroidal shapes, conicalshapes, frusta conical shapes, pyramidal shapes, both as solid andhollow constructs and combination thereof).

[0066] Alternatively, another method to make shaped foamed parts is touse a cold finger (a metal part whose surface represents the inside ofthe part we want to fabricate). The cold finger is dipped into a mixtureof polymer in an appropriate solvent and removed. This is much likedipping an ice cream pop into warm chocolate that freezes to a hard,cold skin, or dipping a form into a latex of rubber to form gloves orcondoms. The thickness and morphology of the foam produced are afunction of the temperature, dwell time and withdrawal rate of the coldfinger in the mixture. Longer dwell, colder finger and slower withdrawalwill produce a thicker coating. After withdrawal, the cold finger isplaced on a fixture of large thermal mass that is in contact with therefrigerated tray of the lyophilizer. From this point the primary andsecondary drying processes are as described above. This method isparticularly well suited to making tubes, branched tubular structures orsleeves that may be shaped to fit devices or portions of an animal'sanatomy (for repair, regeneration or augmentation of tissue).

[0067] Additionally, the polymer solution can be solidified with variousinserts incorporated with the solution such as films, scrims, woven,nonwoven, knitted or braided textile structures. Additionally, thesolution can be prepared in association with another structure such anorthopedic implant (e.g. screws, pins, nails, and plates) or vascular orbranched tubular construct (as a scaffold for a vascularized or ductedorgan). These inserts will be made of at least one biocompatiblematerial and may be non-absorbable, absorbable or a combination thereof.

[0068] The polymer solution in a mold undergoes directional coolingthrough the wall of the mold that is in contact with the freeze dryershelf, which is subjected to a thermal cycle. The mold and its surfacecan be made from virtually any material that does not interfere with thepolymer-solvent system, though it is preferred to have a highlyconducting material. The heat transfer front moves upwards from thelyophilizer shelf through the mold wall into the polymer solution. Theinstant the temperature of the mixture goes below the gellation and/orfreezing point the mixture also phase separates.

[0069] The morphology of this phase separated system is locked in placeduring the freezing step of the lyophilization process and the creationof the open pores is initiated by the onset of vacuum drying resultingin the sublimation of the solvent. However, the mixture in container ormold that is cooled from a heat sink will solidify prior to completelyfreezing. Although the mixture may appear solid, initially there appearsto be some residual solvent associated with the polymer that has notcystallized. It is theorized, but in no way limiting the presentinvention, that a freezing front moves through the mixture from the heatsink to complete the solidification after the mixture has apparentlysolidified. The material in front of the freezing front at a given timewill not be as cold as the material behind the front and will not be ina completely frozen state.

[0070] We have discovered that if a vacuum is applied to the apparentlysolid polymer-solvent mixture immediately after it appears to solidify,a foam with a gradient structure having variable pore size and structureas well as channels can be created. Therefore, timing of the onset ofthe sublimation process (by pressure reduction i.e. vacuum drying) is acritical step in the process to create transitions in the structure. Thetiming of the onset of sublimation will be affected by the thickness ofthe foam being made, concentration of the solution, rate of heattransfer, and directionalities of the heat transfer. Those skilled inthe art will appreciate that this process can be monitored andcharacterized for specific polymer-solvent systems by usingthermocouples and monitoring the heat transfer rates of the foams atvarious depths and locations in the device being foamed. By controllingthe sublimation process, structures with a gradient in pore morphologyand anisotropy may be created. This process can lead to the creation ofmicrostructures that mimic tissues such as cartilage, bone and skin. Forexample, channels will generally be formed if a vacuum is pulledimmediately after the solution apparently solidifies. However, if thesame solution is allowed to solidify further the foam will have largerpores closer to the surface where the vacuum is being drawn (oppositethe heat sink) and smaller pores closer to the heat sink.

[0071] This process is the antitheses of the prior art process thatfocused on creating foams with a uniform microstructure (randomlyinterconnected pores), whereby the whole solution is completely frozen.And vacuum drying is applied only after a considerable amount of time isgiven for the completion of desired phase separation (U.S. Pat. No.5,755,792 (Brekke); U.S. Pat. No. 5,133,755 (Brekke); U.S. Pat. No.5,716,413 (Walter, et al.); U.S. Pat. No. 5,607,474 (Athanasiou, etal.); U.S. Pat. No. 5,686,091 (Leong, et al.); U.S. Pat. No. 5,677,355(Shalaby, et al.); and European disclosures E0274898 (Hinsch) and EPA594148 (Totakura)).

[0072] Foams with various structures are shown in FIGS. 2, 3, and 4. Forexample, as shown in FIG. 3 the orientation of the major axis of thepores may be changed from being in the same plane as the foam to beingoriented perpendicular to the plane of the foam. By way of theory, butin no way limiting the scope of this invention, it is believed that thisin conventional foam processing as the solvent crystallizes a freezingfront moves through the solution solidifying the solution in crystallinelayers parallel to the freezing front. However, if a vacuum is pulledbefore the solution completely freezes, the morphology of the foamresults in pores being formed generally aligned parallel to the vacuumsource. As is illustrated in FIG. 3.

[0073] As can be seen from FIG. 3 the pore size can be varied from asmall pore size generally between about 10 μm and about 60 μm to alarger size of from about 60 μm to about 200 μm in a porous gradientfoam. Again this results from pulling a vacuum on the apparentlysolidified solution before it is completely solidified.

[0074] The polymer concentration in the solution and the cooling ratesare also important parameters in controlling the cell size. Ideally thefoam structure could be created to serve as a template to restore humantissue junctions such as the cartilage to bone junction present injoints. This foam would progress form a small round pores to largercolumn-like (i.e. with a diameter to length ratio of at least 2 to 1)pores. Additionally, the stiffness of the foam can controlled by thefoams structure or blending two different polymers with differentYoung's moduli.

[0075] Foams can also have channels as is illustrated in FIG. 2. Thechannels formed by this process may traverse the thickness of the foamand generally range in diameter from about 30 to about 200 μm indiameter. The channels is generally are at least two times the channel'saverage diameter and preferably are at least four times the channel'saverage diameter and most preferably at least eight times the channel'saverage diameter. The channel size and diameter of course will beselected based on the desired functionality of the channel such as cellinvasion, nutrient diffusion or as a avenue for vascularization.

[0076] One skilled in the art can easily visualize that such adirectionality can be created in any three dimensions by designing anappropriate mold and subjecting the walls of such a mold to differenttemperatures if needed. The following types of gradient structures canbe made by variation in the pore size and/or shape through the thicknesswith a uniform composition: pores of one type and size for a certainthickness followed by another type and size of pores, etc; compositionalgradient with predominantly one composition on one side and another oneon the other with a transition from one entity to the other; a thickskin comprising low porosity of low pore size layer followed by a largepore size region; foams with vertical pores with a spatial organizationthese vertical pores can act as channels for nutrient diffusion themaking of these in 3D molds to create 3D foams with the desiredmicrostructure combinations of compositional and architectural gradient.Generally the foams formed in containers or molds will have a thicknessin the range of from about 0.25 mm to about 100 mm and preferably willhave a thickness of from about 0.5 mm to about 50 mm. Thicker foams canbe made but the lyophilization cycle times may be quite long, the finalfoam structures may be more difficult to control and the residualsolvent content may be higher.

[0077] As previously described the inventive process cycle for producingbiocompatible foam is significantly reduced by performing thesublimation step above the apparent glass transition temperature andbelow the solidification temperature of the mixture (preferably justbelow the solidification temperature). The combined cycle time of(freezing+primary drying+secondary drying) is much faster than isdescribed in the prior art. For example, the combined cycle foraliphatic polyesters using volatile solvents is generally less than 72hours, preferably less than 48 hours, more preferably less than 24 hoursand most preferably less than 10 hours. In fact the combined cycle canbe performed with some aliphatic polyesters in less than 3 hrs for foamsof thickness 1 mm or less; less than 6 hrs for foams of thickness around2 mm and less than 9 hrs for foams of thickness around 3 mm. Comparethis with prior art which is typically 72 hours or greater. The residualsolvent concentrations in these foams made by this process will be verylow. As described for aliphatic polyesters foams made using 1,4-dioxaneas a solvent the residual concentration of 1,4-dioxane was less than 10ppm (parts per million) more preferably less than 1 ppm and mostpreferably less than 100 ppb (parts per billion).

[0078] One skilled in the art can easily visualize that such adirectionality can be created in any three-dimensions by designing anappropriate mold and subjecting the walls of such a mold to differenttemperatures if needed. The following types of gradient structures canbe made by this invention

[0079] 1. variation in the Pore Size and/or Shape Through the Thicknesswith a Uniform Composition,

[0080] 2. pores of one type and size for a certain thickness followed byanother type and size of pores, etc

[0081] 3. compositional gradient with predominantly one compostion onone side and another composition on the other with a transition from oneentity to the other

[0082] 4. a thick skin comprising low porosity of low pore size layerfollowed by a large pore size region

[0083] 5. foams with vertical pores with a spatial organization . . .these vertical pores can act as channels for nutrient diffusion

[0084] 6. the making of these in three-dimensional molds to createthree-dimensional foams with the desired microstructure.

[0085] 7. combinations of compositional and architectural gradient

[0086] Additionally, various proteins (including short chain peptides),growth agents, chemotatic agents and therapeutic agents (antibiotics,analgesics, anti-inflammatories, anti-rejection (e.g.immunosuppressants) and anticancer drugs), or ceramic particles can beadded to the foams during processing, adsorbed onto the surface or backfilled into the foams after the foams are made. For example, the poresof the foam may be partially or completely filled with biocompatibleresorbable synthetic polymers or biopolymers (such as collagen orelastin) or biocompatible ceramic materials (such as hydroxyapatite) andcombinations thereof (that may or may not contain materials that promotetissue growth through the device). Suitable materials include but arenot limited to autograft, allograft, or xenograft bone, bone marrow,morphogenic proteins (BMP's), epidermal growth factor (EGF), fibroblastgrowth factor (FgF), platelet derived growth factor (PDGF), insulinderived growth factor (IGF-I and IGF-II), transforming growth factors(TGF-β), vascular endothelial growth factor (VEGF) or otherosteoinductive or osteoconductive materials known in the art.Biopolymers could also be used as conductive or chemotactic materials,or as delivery vehicles for growth factors. Examples could berecombinant or animal derived collagen or elastin or hyaluronic acid.Bioactive coatings or surface treatments could also be attached to thesurface of the materials. For example, bioactive peptide sequences(RGD's) could be attached to facilitate protein adsorption andsubsequent cell tissue attachment. Therapeutic agents may also bedelivered with these foams.

[0087] In another embodiment of the present invention, the polymers andblends that are used to form the foam can contain therapeutic agents. Toform these foams, the previously described polymer would be mixed with atherapeutic agent prior to forming the foam or loaded into the foamafter it is formed. The variety of different therapeutic agents that canbe used in conjunction with the foams of the present invention is vast.In general, therapeutic agents which may be administered via thepharmaceutical compositions of the invention include, withoutlimitation: antiinfectives such as antibiotics and antiviral agents;chemotherapeutic agents (i.e. anticancer agents); anti-rejection agents;analgesics and analgesic combinations; anti-inflammatory agents;hormones such as steroids; growth factors (bone morphogenic proteins(i.e. BMP's 1-7), bone morphogenic-like proteins (i.e. GFD-5, GFD-7 andGFD-8), epidermal growth factor (EGF), fibroblast growth factor (i.e.FGF 1-9), platelet derived growth factor (PDGF), insulin like growthfactor (IGF-I and IGF-II), transforming growth factors (i.e. TGF-βI-III), vascular endothelial growth factor (VEGF)); and other naturallyderived or genetically engineered proteins, polysaccharides,glycoproteins, or lipoproteins. These growth factors are described inThe Cellular and Molecular Basis of Bone Formation and Repair by VickiRosen and R. Scott Thies, published by R. G. Landes Company herebyincorporated herein by reference.

[0088] Foams containing bio-active materials may be formulated by mixingone or more therapeutic agents with the polymer used to make the foam orwith the solvent or with the polymer-solvent mixture and foamed.Alternatively, a therapeutic agent could be coated on to the foampreferably with a pharmaceutically acceptable carrier.

[0089] Any pharmaceutical carrier can be used that does not dissolve thefoam. The therapeutic agents, may be present as a liquid, a finelydivided solid, or any other appropriate physical form. Typically, butoptionally, the matrix will include one or more additives, such asdiluents, carriers, excipients, stabilizers or the like.

[0090] The amount of therapeutic agent will depend on the particulardrug being employed and medical condition being treated. Typically, theamount of drug represents about 0.001 percent to about 70 percent, moretypically about 0.001 percent to about 50 percent, most typically about0.001 percent to about 20 percent by weight of the matrix. The quantityand type of polymer incorporated into the drug delivery matrix will varydepending on the release profile desired and the amount of drugemployed.

[0091] Upon contact with body fluids the drug will be released. If thedrug is incorporated into the foam then as the foam undergoes gradualdegradation (mainly through hydrolysis) the drug will be released. Thiscan result in prolonged delivery (over, say 1 to 5,000 hours, preferably2 to 800 hours) of effective amounts (say, 0.0001 mg/kg/hour to 10mg/kg/hour) of the drug. This dosage form can be administered as isnecessary depending on the subject being treated, the severity of theaffliction, the judgment of the prescribing physician, and the like.Following this or similar procedures, those skilled in the art will beable to prepare a variety of formulations.

[0092] The foam may also serve as a scaffold for the engineering oftissue. The porous gradient structure would be conducive to growth ofcells. As outlined in previous patents (Vacanti, U.S. Pat. No.5,770,417), cells can be harvested from a patient (before or duringsurgery to repair the tissue) and the cells can be processed understerile conditions to provide a specific cell type (i.e., pluripotentcells, stem cells or precursor cells such as the mesenchymal stem cellsdescribed in Caplan, U.S. Pat. No. 5,486,359, etc.). Suitable cell thatmay be contacted or seeded into the foam scaffolds include but are notlimited to myocytes, adipocytes, fibromyoblasts, ectodermal cell, musclecells, osteoblast (i.e. bone cells), chondrocyte (i.e. cartilage cells),endothelial cells, fibroblast, pancreatic cells, hepatocyte, bile ductcells, bone marrow cells, neural cells, genitourinary cells (includingnephritic cells) and combinations thereof. Various cellular strategiescould be used with these scaffolds (i.e., autogenous, allogenic,xenogeneic cells etc.). The cells could also contain inserted DNAencoding a protein that could stimulate the attachment, proliferation ordifferentiation of tissue. The foam would be placed in cell culture andthe cells seeded onto or into the structure. The foam would bemaintained in a sterile environment and then implanted into the donorpatient once the cells have invaded the microstructure of the device.The in vitro seeding of cells could provide for a more rapid developmentand differentiation process for the tissue. It is clear that cellulardifferentiation and the creation of tissue specific extracellular matrixis critical for the tissue engineering of a functional implant.

[0093] The option for seeding different cell types into the differentpore structures would be available to investigators. Schaufer et al.,have demonstrated that different cell types (i.e. stromal cells andchondrocytes) can be cultured on different structures. The structurescan be combined after a short period of time and the entire structurecan be placed back in cell culture so a biphasic tissue structure can begenerated for implantation. A gradient structure would also allow forco-cultured tissue scaffolds to be generated. (Schaefer, D. et al.).Additionally, radio-opaque markers may be added to the foams to allowimaging after implantation. After a defined period of in vitrodevelopment (for example 3 weeks), the tissue engineered implant wouldbe harvested and implanted into the patient. If an acellular strategy ispursued, then the sterile acellular scaffolds would be used to replacedamaged or traumatized tissue.

[0094] The foam scaffolds of the present invention may be sterilizedusing conventional sterilization process such as radiation basedsterilization (i.e. gamma-ray), chemical based sterilization (ethyleneoxide) or other appropriate procedures. Preferably the sterilizationprocess will be with ethylene oxide at a temperature between 52-55° C.for a time of 8 hours or less. After sterilization the foam scaffoldsmay be packaged in an appropriate sterilize moisture resistant packagefor shipment and use in hospitals and other health care facilities.

[0095] In another embodiment of the present invention the foam may havea fibrous fabric fused to the top or bottom surface. This way, surfaceproperties of the structure can be controlled such as porosity,permeability, degradation rate and mechanical properties. The fibrousfabric can be produced via an electrostatic spinning process in which afibrous layer can be built up on a lyophilized foam surface. Theelectrostatic spinning process is a method that has been used to makefibers. Electrostatic spinning of many polymeric materials has beendescribed in the following patents and articles: U.S. Pat. No.4,522,709; U.S. Pat. No. 5,024,789; U.S. Pat. No. No. 5,311,884; U.S.Pat. No. 5,522,879; “Nanometer diameter fibers of polymer, produced byelectrospinning,” Nanotechnology 1 (1996) 216-233; “Structure andmorphology of small diameter electrospun aramid fibres,” Polymer Inter.,36 (1995) 195-201, “Carbon nanofiber from polyacrylonitrile andmesophase pitch,” Journal of Advanced Materials, Vol. 3, No. 1, (1999)36-41 which are hereby incorporated herein by reference. This processmay also be used with the aliphatic polyesters previously described inthis application using appropriate solvents. The solvents used in thefoaming process may also be used for electrostatic spinning.

[0096] In this process, an electrical force is applied to the polymericsolution that overcomes the surface tension of the solution, forming acharged jet. This jet of solution is ejected, dried and solidified ontoa grounded substrate. By controlling the spinning conditions, theresulting fibers can range from about 0.1 μm to about 20 μm andpreferably will range from about 0.3 μm to about 5.0 μm. The substratesurface in this case would be a lyophilized foam. Two examples of such aconstruct, with a fibrous layer on top of a foam layer, are shown inFIGS. 12 and 13. These layered structures may be made of at least onebiocompatible material and may be non-absorbable, absorbable or acombination thereof.

[0097] The composition, thickness and porosity of the fibrous layer maybe controlled to provide the desired mechanical and biologicalcharacteristics. For example the bioabsorption rate of the fibrous layermay be selected to provide a longer or shorter bioabsorption profile ascompared to the underlying foam layer. Additionally, fibrous layer mayprovide greater structural integrity to the composite so that mechanicalforce may be applied to the fibrous side of the structure. For examplethe fibrous layer could allow the use of sutures, staples or variousfixation devices to hold the composite in place. As a general guidelinebut in no way limiting the scope of the invention it is currentlypreferred that the fibrous layer comprise a layer of from about 1 micronto about 1000 microns in thickness.

[0098] One possible application of a foam structure with a fibroussurface layer is as a vascular graft. The layers in such a structurewould form concentric cylinders, and the fibrous layer would provide adecreased permeability. The lyophilized foam structure could beoptimized for cell infiltration either on the adventitial surface bysmooth muscle cells or fibroblasts, or on the luminal surface byendothelial cells. The fibrous fabric layer would serve as a barrier toblood leakage, and would enhance the mechanical properties of thestructure. Additionally, the fibrous layer would serve as a barrier tomultiplying smooth muscle cells and would assist in the prevention ofintimal hyperplasia.

[0099] Another possible application of such a hybrid foam/fabricstructure is as a scaffold for breast regeneration. The fibrous layerwould provide increased mechanical integrity, and would maintain theoriginal shape of the scaffold. When a biopsy is taken or when a tumoris removed, the cavity could be filled with such a fibrous coated foamscaffold, which may or may not be injected with cells or bioactiveagents.

[0100] Another possible application of such a foam/fabric structure isas a scaffold for meniscal tissue engineering. In this case, a wedgeshaped implant could be made consisting of a fibrous coatingencapsulating a lyophilized foam center. The cells could be injectedinto the center of the implant through the fibrous layer. This fabricsurface layer would allow the diffusion of nutrients and waste productswhile limiting the migration of the cells out of the scaffold.

[0101] The following examples are illustrative of the principles andpractice of this invention, although not limited thereto. Numerousadditional embodiments within the scope and spirit of the invention willbecome apparent to those skilled in the art.

EXAMPLES

[0102] In the examples which follow, the polymers and monomers werecharacterized for chemical composition and purity (NMR, FT-IR), thermalanalysis (DSC), molecular weight (inherent viscosity), and baseline andin vitro mechanical properties (Instron stress/strain).

[0103]¹H NMR was performed on a 300 MHz NMR using CDCl₃ or HFAD(hexafluoroacetone sesqua deutrium oxide) as a solvent. Thermal analysisof segmented polymers and monomers was performed on a Dupont 912Differential Scanning Calorimeter (DSC). Inherent viscosities (I.V.,dL/g) of the polymers and copolymers were measured using a 50 boreCannon-Ubbelhode dilution viscometer immersed in a thermostaticallycontrolled water bath at 25° C. utilizing chloroform orhexafluoroisopropanol (HFIP) as the solvent at a concentration of 0.1g/dL.

[0104] In these examples certain abbreviations are usde such as PCL toindicate polymerized ε-caprolactone, PGA to indicate polymerizedglycolide, PLA to indicate polymerized (L)lactide. Additionally, thepercentages in front of the copolymer indicates the respective molepercentages of each constituent.

Example 1

[0105] Preparation of a Foam with Random Microstructure (no PreferredArchitecture)

[0106] Step A. Preparing 5% wt./wt. Homogeneous Solution of 35/65PCL/PGA in 1,4-Dioxane

[0107] A 5% wt./wt. polymer solution is prepared by dissolving 1 part of35/65 PCL/PGA with 19 parts of the solvent-1,4-dioxane. The 35/65PCL/PGA copolymer was made substantially as described in Example 8. Thesolution is prepared in a flask with a magnetic stir bar. For thecopolymer to dissolve completely, it is recommended that the mixture isgently heated to 60±5° C. and continuously stirred for a minimum of 4hours but not exceeding 8 hours. A clear homogeneous solution is thenobtained by filtering the solution through an extra coarse porosityfilter (Pyrex brand extraction thimble with fritted disc) using drynitrogen to help in the filtration of this viscous solution.

[0108] Step B. Lyophilization

[0109] A laboratory scale lyophilizer—Freezemobile 6 of VIRTIS was usedin this experiment. The freeze dryer is powered up and the shelf chamberis maintained at 200C under dry nitrogen for approximately 30 minutes.Thermocouples to monitor the shelf temperature are attached formonitoring. Carefully fill the homogeneous polymer solution prepared inStep A. into the molds just before the actual start of the cycle. Aglass mold was used in this example but a mold made of any material thatis inert to 1,4-dioxane; has good heat transfer characteristics; and hasa surface that enables the easy removal of the foam can be used. Theglass mold or dish used in this example weighed 620 grams, was opticalglass 5.5 mm thick, and cylindrical with a 21 cm outer diameter and a19.5 cm inner diameter. The lip height of the dish was 2.5 cm. Next thefollowing steps are followed in a sequence to make a 2 mm thick foam:

[0110] (i). The glass dish with the solution is carefully placed(without tilting) on the shelf of the lyophilizer, which is maintainedat 20° C. The cycle is started and the shelf temperature is held at 20°C. for 30 minutes for thermal conditioning.

[0111] (ii). The solution is then cooled to −5° C. by cooling the shelfto −5° C.

[0112] (iii). After 60 minutes of freezing at −5° C., a vacuum isapplied to initiate primary drying of the dioxane by sublimation. Onehour of primary drying under vacuum at −5° C. is needed to remove mostof the solvent. At the end of this drying stage typically the vacuumlevel reached about 50 mTorr or less.

[0113] (iv). Next, secondary drying under a 50 mTorr vacuum or less wasdone in two stages to remove the adsorbed dioxane. In the first stage,the shelf temperature was raised to 5° C. and held at that temperaturefor 1 hour. At the end of the first stage the second stage of drying wasbegun. In the second stage of drying, the shelf temperature was raisedto 20° C. and held at that temperature for 1 hour.

[0114] (v). At the end of the second stage, the lyophilizer is broughtto room temperature and the vacuum is broken with nitrogen. The chamberis purged with dry nitrogen for approximately 30 minutes before openingthe door.

[0115] The steps described above are suitable for making foams that areabout 2 mm thick or less. As one skilled in the art would know, theconditions described herein are typical and operating ranges depend onseveral factors e.g.: concentration of the solution; polymer molecularweights and compositions; volume of the solution; mold parameters;machine variables like cooling rate, heating rates; and the like. FIG. 1shows a SEM of a cross section of the foam produced following theprocess set forth in this example. Note the random microstructure (not apreferred architecture) of this foam.

Example 2

[0116] Preparation of a Foam with Vertical Channels

[0117] This example describes the making of a 35/65 PCL/PGA foam withvertical channels that would provide pathways for nutrient transport andguided tissue regeneration.

[0118] We used a FTS Dura Dry Freeze dryer with computer control anddata monitoring system to make this foam. First step in the preparationof this foam was to generate a homogeneous solution. A 10% wt./wt.homogeneous solution of 35/65 PCL/PGA was made in a manner similar tothat described in Example 1, Step A. The polymer solution was carefullyfilled into a dish just before the actual start of the cycle. The dishweighed 620 grams, was optical glass 5.5 mm thick, and cylindrical witha 21 cm outer diameter and a 19.5 cm inner diameter. The lip height ofthe dish was 2.5 cm. Next the following steps are followed in sequenceto make a 2 mm thick foam with the desired architecture:

[0119] (i). The solution filled dish was placed on the freeze dryershelf that was precooled to −17° C . The cycle was started and the shelftemperature was held at −17° C. for 15 minutes quenching the polymersolution.

[0120] (ii). After 15 minutes of quenching to −17° C., a vacuum wasapplied to initiate primary drying of the dioxane by sublimation andheld at 100 milliTorr for one hour.

[0121] (iii). Next, secondary drying was done at 5° C. for one hour andat 20° C. for one hour. At each temperature the vacuum level wasmaintained at 20 mTorr.

[0122] (iv). At the end of the second stage, the lyophilizer was broughtto room temperature and the vacuum was broken with nitrogen. The chamberwas purged with dry nitrogen for approximately 30 minutes before openingthe door.

[0123]FIG. 2 is a SEM picture that shows a cross section of the foamwith vertical channels. These channels run through the thickness of thefoam.

Example 3

[0124] Architecturally Gradient Foam

[0125] This example describes the making of a foam that has a gradientin foam morphology as shown in FIG. 3 using a 10% solution of 35/65ε-caprolactone-co-glycolide. The method used to make such a foam issimilar to the description given in Example 2 with one difference. Instep (ii) of the lyophilization process the time for which the solutionis kept at the freezing step is 30 minutes.

[0126]FIG. 3 is a scanning electron micrograph of a cross section ofthis foam. Note the variation in the pore size and pore shape throughthe thickness of the foam.

Example 4

[0127] Transcompositional Foam

[0128] This example describes the making of a foam that has acompositional gradient and not necessarily a morphological gradient.Such a foam is made from polymer solutions that have been made fromphysical mixtures of two or more polymers. This example describes atranscompositional foam made from 35/65 PCL/PGA and 40/60 PCL/PLA

[0129] Step A. Preparing a Solution Mixture of 35/65 PCL/PGA and 40/60PCL/PLA in 1,4-Dioxane

[0130] In the preferred method the two separate solutions are firstprepared (a) a 10% wt/wt polymer solution of 35/65 PCL/PGA and (b) a 10%wt/wt 40/60 PCL/PLA. Once these solutions are prepared as described inExample 1, equal parts of each solution was poured into one mixingflask. The polymers used to make these solutions are described inExamples 8 and 9. A homogeneous solution of this physical mixture wasobtained by gently heating to 60±5° C. and continuously stirring using amagnetic stir bar for approximately 2 hours.

[0131] Step B. Lyophilization Cycle

[0132] We used an FTS Dura Dry Freeze dryer with computer control anddata monitoring system to make this foam. The first step in thepreparation of such a foam was to generate a homogeneous solution asdescribed in Step A. The solution was carefully filled into a dish justbefore the actual start of the cycle. The cylindrical glass dish weighed117 grams, was optical glass 2.5 mm thick and cylindrical with a 100 mmouter diameter and a 95 mm inner diameter. The lip height of the dishwas 50 mm. Next the following steps were followed in sequence to make a25 mm thick foam with the transcompositional gradient:

[0133] (i). The solution filled dish was placed on the freeze dryershelf and the solution conditioned at 20° C. for 30 minutes. The cyclewas started and the shelf temperature was set to −5° C. with aprogrammed cooling rate of 0.5° C./min.

[0134] (ii). The solution was held at the freezing condition (−5° C.)for 5 hours.

[0135] (iii). Vacuum was applied to initiate primary drying of thedioxane by sublimation and held at 100 milliTorr for 5 hours.

[0136] (iv). Next, secondary drying was done at 5° C. for 5 hours and at20° C. for 10 hours. At each temperature the vacuum level was maintainedat 20 mTorr.

[0137] (v). At the end of the second stage, the lyophilizer was broughtto room temperature and the vacuum was broken with nitrogen. The chamberwas purged with dry nitrogen for approximately 30 minutes before openingthe door.

[0138] The foam has a gradient in chemical composition which is evidentfrom a close scrutiny of the foam wall morphology as shown in FIGS. 4, 5and 6. The gradient in the chemical composition was further supported byNMR data as detailed below:

[0139] Foam sample produced by the above method and which wasapproximately 25 mm thick was characterized for mole % composition. Thefoam sample is composed of a physical blend of PCL/PLA and PCL/PGA.Slices of the foam sample were prepared and analyzed to confirm that thematerial was a compositional gradient. The sample slices were identifiedas 1) foam IA (top slice), 2) foam IB (top middle slice), 3) foam IC(bottom middle slice), 4) foam ID (bottom slice). The NMR samplepreparation consisted -1390 of dissolving a 5 mg of material into 300 μLhexafluoroacetone sesqua deutrium oxide (HFAD) and then diluting with300 μL of C₆D₆. 1 H NMR Results: Mole % Composition Sample ID PLA PGAPCL Foam IA 47.2 12.4 40.5 Foam IB 12.3 51.3 36.5 Foam IC 7.7 56.5 35.8Foam ID 7.8 56.3 35.8

[0140] The NMR results indicate that the foam samples have a gradientwith respect to composition. The top layer of the foam is high in PLAconcentration (47 mole %), whereas the bottom layer of the foam is highin PGA concentration (56 mole %). These results suggest that the PCL/PGAcopolymer and the PCL/PLA copolymer have differences in their phaseseparation behaviors during the freezing step and formed a uniquecompositionally gradient foam.

Example 5

[0141] Transstructural Foam

[0142] This example describes the making of a foam that has acompositional and structural gradient and not necessarily amorphological gradient. Such a foam is made from polymer solutions thathave been made by physical mixtures of two or more polymers. Thisexample describes a transcompositional foam made from 35/65 PCL/PLA (asdescribed in Example 9) and 95/5 PLA/PCL (a random copolymer with an IVof 1.8 in HFIP measured as described herein). Note, 35/65 PCL/PLA is asoft elastomeric copolymer while 95/5 PLA/PCL is a relatively stiffcopolymer. The combination of the two provides a compositional as wellas structural gradient. This foam is made using the steps outlined inExample 4 starting from a homogeneous 50/50 physical mixture of a 10%wt./wt. solution of 35/65 PCL/PLA and 10% wt./wt. Solution of 95/5PLA/PCL in 1,4 dioxane. Such a transcompositional foam will provide agood template for tissue junctions such as bone-cartilage interfaces.

Example 6

[0143] Cell Culture and Differentiation Data

[0144] Films made from 95/5 PLA/PGA, 90/10 PGA/PLA, 95/5 PLA/PCL, 75/25PGA/PCL and 40/60 PCL/PLA were tested. Tissue culture polystyrene (TCPS)was used as a positive control for all the assays. Before testing,polymer discs were positioned at the bottom of a 24-well ultralowcluster dish and were pre-wetted in growth media for 20 min.

[0145] The 95/5 PLA/PGA copolymer used in this example was a randomcopolymer with an IV of 1.76 as determined in HFIP at 25° C., which iscurrently used in Panacryl™ suture (Ethicon Inc., Somerville, N.J.). The90/10 PGA/PLA copolymer was a random copolymer with an IV of 1.74 asdetermined in HFIP at 25° C., which is currently used in Vicyl™ suture(Ethicon Inc., Somerville, N.J.). The 95/5 PLA/PCL polymer was made asdescribed in Example 10, with an IV of 2.1 as determined in HFIP at 25°C. The 75/25 PG/PCL copolymer is a segmented block copolymer with an IVof 1.85 and is described in U.S. Pat. No. 5,133,739 this copolymer iscurrently used in Monocryl™ sutures (Ethicon Inc., Somerville, N.J.).The 40/60 PCL/PLA copolymer used in this Example was made as describedin Example 9 and had an IV of 1.44.

[0146] Cell Attachment and Proliferation:

[0147] Cells were seeded at 40,000/well in 24-well ultralow clusterdishes (Corning) containing the polymers. The ultralow cluster dishes 5are coated with a layer of hydrogel polymer, which retards protein andcell adhesion to the wells. Cell attachment to the biopolymers wasdetermined following 24 hrs of incubation (N=3 for each polymer). Theattached cells were released by trypsinization and the number of cellswas determined using a heamacytometer. Cell proliferation was assessedby determining cell counts at days 3 and 6 following seeding.

[0148] Differentiation Assays:

[0149] Alkaline Phosphatase Activity:

[0150] Alkaline phosphatase activity was determined by a calorimetricassay using p-nitrophenol phosphate substrate (Sigma 104) and followingmanufacturers instruction. Briefly, cells were seeded on the films ormeshes at a density of 40,000 cells/well and incubated for 1, 6, 14, and21 d. Once cells reached confluence at day 6 they were fed withmineralization medium (growth medium supplemented with 10 mMβ-glycerophosphate, 50 μg/ml ascorbic acid). Alkaline phosphataseactivity was determined in cell homogenates (0.05% Triton X-100) at theabove time points. The quantity of protein in cell extracts wasdetermined by micro BCA reagent from Pierce. Primary rat osteoblastscultured on films and meshes were also stained for membrane-boundalkaline phosphatase using a histochemical staining kit (Sigma). For allthe films and meshes three samples per group were tested.

[0151] Osteocalin ELISA:

[0152] Osteocalcin secreted into the medium by osteoblasts cultured onvarious films was quantified by ELISA (Osteocalcin ELISA kit, BiomedicalTechnologies Inc, Boston). Aliquots of media from the wells containingthe polymer films were lyophilized prior to measurements of this proteinby ELISA. Three samples for each polymer were tested and the ELISA wasrepeated twice.

[0153] Von Kossa Staining

[0154] Three samples for each polymer were stained for mineralizedtissue using Von Kossa silver nitrate staining.

[0155] Expression of Alkaline Phoshatase and Osteocalcin mRNAs

[0156] The expression of alkaline phosphatase and osteocalcin mRNAs incells was assessed by semi-quantitative reverse transcriptase polymerasechain reaction (RT-PCR) using RNA extracted from cells cultured for 21 don the films. Seven days after seeding, the culture media was replacedwith mineralization media (3 mM β-glycerophosphate and 50 μg/ml ofascorbic acid were added). The cells were cultured for additional 2weeks, for a total period of 3 weeks. Total RNA was extracted from foursamples per group using a RNeasy mini kit provided by Qiagen. Thequality and amount of total RNA was measured for each polymer group.Total RNA was reverse transcribed to obtain cDNA using a reversetranscriptase reaction (Superscript II, Gibco). The cDNAs forosteocalcin, alkaline phosphatase, andGlyceraldehyde-3-phosphate-dehydrogenase (GAPDH) were amplified using aPCR protocol described previously (GIBCO BRL manufacturers instruction).The primer sequences (Table I) for osteocalcin, alkaline phosphatase,and GAPDH were obtained using the FASTA program (Genetic Computer Group,Madison, Wis.). Preliminary studies were also conducted to optimize thenumber of PCR cycles for each primer (Table II), and to determine therange of RNA, which exhibits proportionality to cDNA. The PCR productswere electrophoreses on 1% (wt) agarose gels containing ethidiumbromide. The gels were photographed under UV light and were evaluated bydensitometry for the expression of osteocalcin and alkaline phosphatasemRNAs relative to GAPDH.

[0157] Statistical Anlysis

[0158] Analysis of variance (ANOVA) with Tukey post hoc comparisons wasused to assess levels of significance for all the assays. TABLE IPrimers used in RT-PCR Size Gene Species Forward primer Reverse primer(bp) Alka- Rat 5′ 5′ 379 line ATCGCCTATCAGCT GCAAGAAGAAGCCT phos-AATGCAC TTGGG phatase Osteo- Rat/ 5′CAACCCCAATTG 5′ 339 calcin HumanTGACGAGC TGGTGCGATCCATC ACACAG GAPDH Mouse/ 5′ACCACAGTCCAT5′TCCACCACCCTG 452 Human/ GCCATCAC TTGCTGTA Rat

[0159] TABLE II PCR optimization cycles Gene cDNA (μl) Cycles Alkaline 125 phosphatase Osteocalcin 1 35 GAPDH 1 23

[0160] Results

[0161] Cell Attachment and Proliferation on Bioresorbable Polymers:

[0162] No observable difference in cell morphology was evident betweenthe various polymer films and as compared to TCPS. Cell attachment tothe various biopolymer films was equivalent to TCPS following 24 h ofincubation. At day 3, cells proliferated well on all films with theexception of 40/60 PCL/PLA, where proliferation was 60% relative toTCPS. Furthermore, 95/5 PLA/PGA and 90/10 PGA/PLA films supported asignificantly (p<0.05) higher degree of cell proliferation compared toTCPS and 40/60 PCL/PLA (FIG. 7A).

[0163] Differentiation Assay:

[0164] Alkaline Phosphatase Enzyme Activity:

[0165] The profile for alkaline phosphatase activity expressed byosteoblasts cultured on 95/5 PLA/PGA, 90/10 PGA/PLA and 95/5 PLA/PCLfilms was similar to the profile observed on TCPS. Alkaline phosphatasespecific activities were significantly (p<0.05) elevated for osteoblastscultured on 40/60 PCL/PLA and 75/25 PGA/PCL films at days 14 and 21,respectively, compared to other films and TCPS (FIG. 7B).

[0166] Expression of Alkaline Phosphatase and Osteocalcin mRNA:

[0167] The expression of mRNAs for alkaline phosphatase, osteocalcin,and GAPDH for osteoblasts cultured on the 95/5 PLA /PGA, 40/60 PLA/PCL,95/5 PLA/PCL films, and TCPS were evaluated by densitometry. The resultsare depicted in FIG. 7C. It should be noted that the data in FIG. 7B isat best semi-quantitative. Nevertheless, the data suggests that 40/60PCL/PLA film supported significantly (p<0.05) higher levels ofosteocalcin expression compared to TCPS. The rest of the polymersurfaces were equivalent to TCPS for both osteocalcin and AP mRNAsexpression.

[0168] Conclusions

[0169] No major differences were observed with respect to cellattachment and proliferation between the different bioresorbable filmsor meshes tested following 6 days of incubation. Furthermore, theresults indicate that differences between these materials were moreobvious with respect to their differentiation characteristics. Cellscultured on 40/60 PCL/PLA film showed enhanced alkaline phosphataseactivity and osteocalcin mRNA expression compared to other films andTCPS following 14 and 21 days of incubation, respectively.

[0170] References that may be referred to for a more completeunderstanding of this techniques include, M. A. Aronow, L. C.Gerstenfeld, T. A. Owen, M. S. Tassinari, G. S. Stein and J. B. Lian:“Factors that promote progressive development of the osteoblastphenotype in cultured fetal rat calvaria cells: Journal of CellularPhysiology, 143: 213-221 (1990) and Stein, G. S., Lian, J. B., and Owen,t. A. “Relationship of cell growth to the regulation of tissue-specificgene expression during osteoblast differentiation” FASEB, 4, 3111-3123(1990).

Example 7

[0171] In Vivo Study of Foam Blend in Swine Dermal Wound Healing Model

[0172] This example describes the results of implanting a 1 mm, 0.5 mmthickness foam tissue scaffolding in a swine full thickness excisionalwound model. The foam tissue scaffold was made from a blend of 40/60ε-caprolactone-co-lactide made as described in Example 8 and 35/65ε-caprolactone-co-glycolide described in Example 9. These polymers wereblended together and formed into 1 mm and 0.5 mm foams substantially asdescribed in Example 3 (except that the cooling rate was 2.5° C. perminute and it was cooled only to −5° C.). Scanning electron micrographsof a 0.5 mm foam are presented in FIGS. 9A, 9B and 9C. The two thickness(0.5 mm and 1 mm) of foams were then tested in the wound excisionalmodel with and without PDGF being provided. The resulting four differentsamples were then evaluated.

[0173] A blinded histologic evaluation was performed on 48 fullthickness excisional wounds from four pigs (12 sites per animal)explanted at 8 days following wounding. The assessment was performed onH&E stained slides. During the histologic assessment, the followingparameters were ranked/evaluated across the specimen set 1) cellularinvasion of the matrix qualitative and quantitative assessments 2)infiltration of polymorphonuclear leukoctyes (PMNs) into the contactzone (ventral surface) of the matrix, 3) inflammation in the granulationtissue bed below (ventral to) the matrix, 4) reaction of the epidermisto the matrix, and 5) degree of fragmentation of the matrix.

[0174] Animal Husbandry:

[0175] The pigs were housed individually in cages (with a minimum floorarea of 10-sq. ft.) and given identification. All pigs were assigned anindividual animal number. A tag was placed on each individual animalcage listing the animal number, species/strain, surgical date, surgicaltechnique and duration of the experiment and date of euthanasia. Eachanimal was clearly marked with an animal number on the base of the neckusing a permanent marker.

[0176] The animal rooms were maintained at the range of 40 to 70% R.H.and 15 to 24° C. (59.0 to 75.2° F.). The animals were fed with astandard pig chow once per day, but were fasted overnight prior to anyexperimental procedure requiring anesthesia. Water was available adlibitum. A daily light:dark cycle of 12:12 hours was adopted.

[0177] Anesthesia:

[0178] On the initial day of the study, days of evaluation and the dayof necropsy, the animals were restrained and anesthetized with either anintramuscular injection of Tiletamine HCl plus Zolazepam HCl (Telazol®,Fort Dodge Animal Health, Fort Dodge, Iowa 4 mg/ml) and xylazine(Rompun®, Bayer Corporation, Agriculture Division, Animal Health,Shawnee Mission, Kans., 4 mg/ml) or Isoflurane (AErrane® Fort DodgeAnimal Health, Fort Dodge, Iowa) inhalatory anesthesia (5% vol.)administered via a nose cone. When the animal was in the surgical suite,it was maintained on Isoflurane (AErrane®) inhalatory anesthesia (2%vol.) administered via a nose cone. Food was available after recoveryfrom each procedure.

[0179] Preparation of the Surgical Site:

[0180] One day prior to the surgical procedure, body weights weremeasured and the dorsal region of four pigs were clipped with anelectric clipper equipped with a #40 surgical shaving blade. The shavedskin was then re-shaved closely with shaving cream and a razor and thenrinsed. The shaved skin and entire animal (excluding the head) was thenscrubbed with a surgical scrub brush-sponge with PCMX cleansing solution(Pharmaseal® Scrub Care® Baxter Healthcare Corporation, PharmasealDivision, Valencia, Calif.) and then with HIBICLENS® chlorhexidinegluconate (available from COE Laboratories, Incorporated, Chicago,Ill.). The animal was wiped dry with a sterile towel. Sterile NU-GAUZE*gauze (from Johnson & Johnson Medical Incorporated, Arlington, Tex.) wasplaced over the dorsal surface of each animal and secured withWATERPROOF* tape (available from Johnson & Johnson Medical Incorporated,Arlington, Tex.). The entire torso region of the animal was then wrappedwith Spandage™ elastic stretch bandage (available from Medi-TechInternational Corporation, Brooklyn, N.Y.) to maintain a clean surfaceovernight.

[0181] On the day of surgery, immediately prior to delivering the animalto the surgical suite, the dorsal skin was again scrubbed using asurgical scrub brush-sponge with PCMX cleansing solution (Pharmaseal®Scrub Care®), rinsed and wiped dry using a sterile towel, as performedon the previous day. The animals were placed prone on the surgical tableand wiped with 70% alcohol and dried with sterile gauze. Using a sterilesurgical marker (availabe from Codman® a division of Johnson & JohnsonProfessional Incorporated, Raynham, Mass.) and an acetate template,marks were made on the dorsal skin according to the desired placement ofeach full-thickness wound.

[0182] Surgical Procedure:

[0183] Following anesthesia, under sterile conditions, twelve (12)full-thickness excisions (1.5×1.5 cm) per animal were made in two rowsparallel to the spinal column on the left and right dorsal regions usinga scalpel blade.

[0184] A pair of scissors and/or scalpel blade was used to aid in theremoval of skin and subcutaneous tissue. Bleeding was controlled by useof a sponge tamponade. Sufficient space was left between wounds to avoidwound-to-wound interference. The excised tissue was measured forthickness using a digital caliper.

[0185] Application of the Treatment and Dressing:

[0186] Each wound was submitted to a prepared, coded treatment regimen(study participants were blinded to all treatments). The primarydressing consisting of the sterile individual test article (1.5×1.5 cmsoaked in sterile saline for 24 hours) was placed into the wound deficitin a predetermined scheme. The secondary dressing, a non-adherent,saline soaked, square of RELEASE* dressing (manufactured by Johnson &Johnson Medical Incorporated, Arlington, Tex.) was placed on top of thetest article. A layer of BIOCLUSIVE* dressing (available from Johnson &Johnson Medical Incorporated, Arlington, Tex.) was sealed over thewounds to keep the wound moist and the dressing in place. Strips ofReston™ (3M Medical-Surgical Division, St. Paul, Minn.) polyurethaneself-adhering foam were placed between the wounds to avoidcross-contamination due to wound fluid leakage, and to protect thewounds from damage and the dressing from displacement. A layer ofNU-GAUZE* gauze was placed on top of the BIOCLUSIVE* dressing andReston™ foam, and was secured with WATERPROOF* tape to protect thedressings. The animals were then dressed with Spandage” elastic net tohelp keep the dressings in place.

[0187] The secondary dressings were removed and replaced daily with afresh piece of saline soaked RELEASE* secondary dressing. The primarydressings (test articles) were not disturbed unless the unit wasdisplaced or pushed out of the wound deficit.

[0188] Post-Operative Care and Clinical Observations:

[0189] After performing the procedures under anesthesia, the animalswere returned to their cages and allowed to recover. The animals weregiven analgesics (buprenorphine hydrochloride [Buprenex Injectable, 0.01mg/kg, im] sold by Reckitt & Colman Products, Hull, England) immediatelypost-surgery and the following day.

[0190] After recovering from anesthesia, the pigs were observed forbehavioral signs of discomfort or pain. No signs of pain were observed.

[0191] Each pig was observed twice daily after the day of surgery todetermine its health status on the basis of general attitude andappearance, food consumption, fecal and urinary excretion and presenceof abnormal discharges.

[0192] Euthanasia:

[0193] At the end of the study (8 days post-wounding), each animal waseuthanized under anesthesia, with an intravenous injection of (1 ml/10pounds body weight) Socumb™ pentobarbital sodium and phenytoin sodiumeuthanasia solution (sold by The Butler Company, Columbus, Ohio) via themarginal ear vein. Following euthanasia, the animals were observed toensure that respiratory function had ceased and there was no palpablecardiac function. A stethoscope facilitated the assessment for the lackof cardiac function.

[0194] Tissue Harvesting:

[0195] Immediately following euthanasia, each wound, together with theunderlying fat and a small portion of surrounding skin was excised. Thetissue was placed in 10% neutral buffered formalin.

[0196] Evaluations:

[0197] Visual Wound Assessment:

[0198] General observations were recorded for days 1-3, includingdisplacement, wound reaction and physical characteristics of thescaffold. Detailed clinical evaluations were performed on days 4-8post-wounding. Assessments were recorded as to the presence/absence(yes=1/no=0) and/or degree (given a score) of the following parameters:

[0199] Dressing Conditions: air exposed, displacement of test article,channeling, communication and moisture content of the RELEASE* secondarydressing(scored as: 4=moist, 3=moist/dry, 2=dry/moist, 1=dry).

[0200] Wound Bed Conditions: moisture content of test article (scoredas: 4=moist, 3=moist/dry, 2=dry/moist, 1=dry), inflammation (scored as:3=severe, 2=moderate, 1=slight, 0=none), reinjury (scored as: 3=severe,2=moderate, 1=slight, 0=none), clots, folliculitis, infection, level oftest article (scored as: 4=super raised, 3=raised, 2=even, 1=depressed),fibrin (scored as: 3=severe, 2=moderate, 1=slight, 0=none), anderythema. Color of the test article was also observed.

[0201] Tissue Processing:

[0202] Excised tissue samples were taken at day eight. The entire woundwas harvested and placed into 10% neutral buffered formalin. The tissuewas prepared for frozen sections. The tissue was trimmed and mountedonto the object holder with Tissue-Tek® OCT Compound (sold by SakuraFinetechnical Company, Limited, Tokyo, Japan) and quickly frozen. Thespecimens were sectioned on the cryostat at 10 μm and stained with afrozen H&E stain.

[0203] Histological Assessments (Day 8 Post-Wounding):

[0204] Histological evaluations for granulation tissue (area and length)and epithelialization were assessed using H&E stained specimens using amagnification of 20-40×. Granulation tissue height was determined bydividing the area by the length.

[0205] Histopathological evaluation of the tissue samples was assessedusing the H&E stained specimens, they were first assessed under 100× to400× magnification.

[0206] Results

[0207] There was cellular invasion into the interstices of the matrix inthe majority of all test sites. In the majority of sites this invasionwas true tissue in-growth comprised of varying populations offibroblasts, macrophages, macrophage giant cells, and endothelial-likecells, there also appeared to be capillary formation. Significantformation of dense fibrous connective tissue layer dorsal to thematrices essentially embedding the matrices in the tissue, was seen atseveral sites for the 0.5 mm foams with and without PDGF. The 1 mmmatrices were either at the surface of the tissue bed or sloughed.Macrophage giant cell formation seemed to be greater in the 0.5 mmversus the 1 mm foam scaffolds. In sites where the 1 mm foam was beingsloughed or partially separated from the underlying granulation tissuethere was death of the invading cells forming masses of pyknotic celldebris.

[0208] Complete incorporation of the matrix into the granulation tissuebed was seen at several sites for the 0.5 mm foam scaffoldings. FIGS. 10and 11 illustrate the incorporation of these matrices into thegranulation tissue bed. FIG. 10 is a dark filed 40× pictomicrograph of atrichrome stained tissue sample. FIG. 11 is a 100× compositephotomicrograph of a trichrome stained sample illustrating the cellularinvasion of a foam containing PDGF. Complete incorporation of thematrices into the granulation tissue bed is evident in both pictures.The dense fibrous tissue above the foam scaffolding is evident in bothpictures. These results indicate the 0.5 mm foams will provide asuitable substrate for the growth of epidermal tissue.

Example 8

[0209] Synthesis of a Random Poly(ε-caprolactone-co-glycolide)

[0210] A random copolymer of ε-caprolactone-glycolide with a 35/65 molarcomposition was synthesized by ring opening polymerization reaction. Themethod of synthesis was essentially the method described in U.S. Pat.No. 5,468,253 in Example 6 (which is hereby incorporated herein byreference). The amount of diethylene glycol initiator added was adjustedto 1.15 mmole/mole of monomer to obtain the following characteristics ofthe dried polymer: The inherent viscosity (I.V.) of the copolymer was1.59 dL/g in hexafluoroisopropanol at 25° C. The molar ratio of PCL/PGAwas found to be 35.5/64.5 by proton NMR with about 0.5% residualmonomer. The glass transition (Tg) and the melting points (Tm) of thecopolymer were found to be −10C, 600C and 126° C. respectively, by DSC.

Example 9

[0211] Synthesis of 40:60 Poly(ε-caprolactone-co-L-lactide) bySequential Addition

[0212] In the glove box, 100

[0213] L (33 μmol) of a 0.33 M stannous octoate solution in toluene, 115μL (1.2 mmol) of diethylene glycol, 24.6 grams (170 mmol) of L-lactide,and 45.7 grams (400 mmol) of ε-caprolactone were transferred into asilanized, flame dried, two neck, 250 mL round bottom flask equippedwith a stainless steel mechanical stirrer and a nitrogen gas blanket.The reaction flask was placed in an oil bath already set at 190° C. andheld there. Meanwhile, in the glove box, 62.0 grams (430 mmol) L-lactidewere transferred into a flame dried, pressure equalizing additionfunnel. The funnel was wrapped with heat tape and attached to the secondneck of the reaction flask. After 6 hours at 190° C., the moltenL-lactide was added to the reaction flask over 5 minutes. The reactionwas continued overnight for a total reaction time of 24 hours at 190° C.The reaction was allowed to cool to room temperature overnight. Thecopolymer was isolated from the reaction flask by freezing in liquidnitrogen and breaking the glass. Any remaining glass fragments wereremoved from the copolymer using a bench grinder. The copolymer wasagain frozen with liquid nitrogen and broken off the mechanical stirringpaddle. The copolymer was ground into a tared glass jar using a WileyMill and allowed to warm to room temperature in a vacuum oven overnight.103.13 grams of 40:60 poly(ε-caprolactone-co-L-lactide) were added to atared aluminum pan and then devolitilized under vacuum at 110° C. for 54hours. 98.7 grams (95.7% by weight) of copolymer were recovered afterdevolitilization. The inherent viscosity was measured and found to be1.1 dL/g CHCl₃ at 25° C. (c=0.1 g/dL). FTIR (cast film from CHCl₃ ontoKBr window, cm⁻¹): 2993, 2944, 2868, 1759, 1456, 1383, 1362, 1184, 1132,1094, 870, and 756. ¹H NMR (400 MHz, HFAD/Benzene, ppm): δ 1.25, 2 broadlines (e); 1.35, 2 lines (f); 1.42, 3 lines; 1.55, 2 lines; 2.22, 3lines; 2.35, 4 broad lines; 4.01, 3 lines; 4.05, 3 lines; 4.2, quartet;5.05, 3 broad lines; 5.15, 4 lines. Polymer composition by ¹H NMR: 41.8%PCL, 57.5% PLA, 0.8% L-lactide, <0.1% s-caprolactone. DSC (20° C./min,first heat): T_(m)=154.8° C., ΔH_(m)=18.3 J/g. GPC (molecular weightsdetermined in THF using poly(methyl methacrylate) standards, daltons):M_(w)=160,000, M_(w)=101,000, PDI=1.6.

Example 10

[0214] Synthesis of 95/5 PLA/PCL Copolymer

[0215] In the glove box, 170 μL (1.8 mmol) of diethylene glycol, 350 μL(115 μmol) of a 0.33 M stannous octoate solution in toluene, 17.1 grams(150 mmol) of ε-caprolactone, and 410.4 grams (2.85 mol) of L-lactidewere placed into a silanized, flame dried, 1000 mL round bottom equippedwith a stainless steel mechanical stirrer and vacuum take off connectorin order to maintain a dry nitrogen gas blanket. The reaction flask wasplaced in an oil bath already heated to 185° C. and then held there for3 hours. The flask was removed from the oil bath and allowed to cooldown to room temperature. The polymer was isolated by wrapping the flaskwith aluminum foil, freezing it in liquid nitrogen, and then grindingaway any adhered glass to the polymer. The copolymer was then ground ina Wiley mill. The ground polymer was vacuum dried at 80° C. for 24hours. 302 grams of copolymer were collected. The inherent viscosity was2.1 dL/g in chloroform [250C, c=0.1 g/dL]. The copolymer composition wasmeasured by proton NMR spectroscopy and found to be 97.2 mole percentPLA and 2.8 mole percent PCL. No residual monomer was detected.

Example 11

[0216] Synthesis of 60/40 PLA/PCL Hybrid Foam/Microfibrous FabricStructure

[0217] Step A. Preparing 14% wt./wt. Homogenous Solution of 60/40PLA/PCL in Trichloroethane

[0218] A 14% wt./wt. Polymer solution is prepared by dissolving 14 parts60/40 PLA/PCL (I.V. =1.34 dl/g) with 86 parts of the solventtrichloroethane (TCE). The solution is prepared in a flask with amagnetic stir bar. For the copolymer to dissolve completely, it isrecommended that the mixture stir overnight at 60 degrees C.

[0219] Step B. Electrostatic Spinning

[0220] The electrostatic spinner was used for this experiment. SpellmanHigh Voltage DC Supply (Model No.: RHR30PN30, Spellman High VoltageElectronics Corporation, Hauppauge, N.Y., USA) was used as high voltagesource. Applied voltage and the speed of mandrel were controlled by theLabview computer software. Distance between the spinneret wasmechanically controlled.

[0221] A flat sheet of lyophilized 60/40 PLA/PCL foam, prepared asdescribed in Example 1, was rolled into a cylinder. The ends weresecured together with several drops of trichloroethane (TCE), thesolvent used in spinning. The foam tube was mounted onto a rotatingconductive mandrel that was grounded. The ends of the mandrel notcovered by the foam substrate were masked with an insulating tape toprevent the attraction of the microfibers to the ends. The solution wasplaced into a spinneret and high voltage was applied to the polymersolution. This experiment was performed at ambient temperature andhumidity.

[0222] The operating conditions during spinning were as follows: Mandrelvoltage 16,000 V Mandrel speed   100 rpm Spinneret to mandrel distance   10 cm Temperature room temperature Humidity ambient humidity

[0223] A layer of approximately 200 um in thickness was deposited on theouter surface of the tube

Example 12

[0224] Chondrocytes Cultured in an Encapsulated Hybrid Foam/FabricStructure Demonstrating the Limitation of Cell Migration

[0225] Step A. Preparation of hybrid 60/40 PLA/PCL Foam/MicrofibrousFabric Scaffolds

[0226] Lyophilized foams made from 60/40 PLA/PCL as described in Example1 were used as substrates during the electrostatic spinning of 60/40PLA/PCL in trichloroethane, as described in detail in Example 11.

[0227] Step B. Chrondrocyte Seeding into the Hybrid 60/40 PLA/PCLFoam/Microfibrous Fabric Scaffold

[0228] Chondrocytes isolated from bovine shoulders were cultured inDulbecco's modified Eagle medium (high glucose) supplemented with 10%fetal calf serum, 10 mM HEPES, 0.1 mM nonessential amino acids, 20 ug/mlstreptomycin, and 0.2S ug/ml amphotericin B.

[0229] 3-D Transmigration Assay:

[0230] This assay is based on cell-populated contracted collagen latticewith a biodegradable polymer scaffold implanted at the center of thecollagen gel. The dermal equivalents (collagen lattice) were fabricatedfrom a mixture of the following: 8 ml of chondrocyte media, 2 ml ofneutralizing medium (DMEM/10% FBS containing 0.1 N NaOH, prepared priorto use), 4 ml of rat tail type I collagen (3.99 mg/ml) obtained fromCollaborative Biomedical Products, and 2 ml of chondrocyte suspension inchondrocyte medium. The above cell/collagen mixture was distributed (2ml) to each well of a 24-well hydrogel-coated plate (Costar, Cat #3473). The final cell concentration was 6×10⁶ cells/ml and the collagenconcentration was 1 mg/ml. The collagen gels was allowed to polymerizeat 37° C. for 1 h. Following gel polymerization, 1.5 ml of chondrocytegrowth medium was applied into each well. The medium was changed everyother day. After 7 days of contraction, wounds at 5 mm in diameter weremade throughout the thickness of the collagen lattice using a disposable5 mm biopsy punch. Tested scaffolds, approximately 5 mm in diameter and2 mm thick, supplied in a wet form, were implanted into a center of thewounded collagen lattice. To assure that the scaffold would not bepushed out from the wound bed during contraction of the collagenlattice, 50 μl of acellular collagen was placed on the top of eachconstruct. Constructs were cultivated in chondrocyte media with mediumbeing changed in a 2-day interval. The contracted gels containing thescaffold were cultured either statically in ultra low cluster dishes orin the bioreactor and collected after 3 weeks.

[0231] Step C. Cell Characterization Within the Scaffolds

[0232] Histology was performed on the scaffolds invaded by cells.Scaffolds were fixed in 10% buffered formalin and were either embeddedin paraffin and sectioned (hybrid foam/microfibrous fabric scaffolds) orcryosectioned (control foam scaffolds). Sections were stained withHematoxylin/eosin (H/E, cell number and morphology), Safranin O (SO;sulfated GAGs) and

[0233] Trichrome (Collagen).

[0234] Quantitation of cell invasion into these scaffolds was determinedby image analysis. Analysis of Variance with Tukey post hoc tests wereperformed on the data.

[0235] Experimental Results

[0236] Quantitative evaluation of H/E sections of constructs culturedfor 3 weeks revealed that chondrocytes invaded both scaffolds. Cellinvasion into the hybrid foam/microfibrous fabric scaffolds, cultured inthe bioreactor, was significantly higher (P<0.05) than into 40/60PCL/PLA foam scaffolds. Also in the statically maintained cultures thecell invasion into the hybrid foam/microfibrous fabric scaffolds wassignificantly (P<0.05) higher than into 40/60 PCL/PLA foam scaffolds.Cell invasion Hybrid cells/mm2 40/60 PCL/PLA foam/microfibrous (3 weeks)foam scaffolds fabric scaffolds Static Cell 50 80 Culture Bioreactor 140200 Cell Culture

[0237] Hybrid foam/microfibrous fabric scaffolds exhibited the highestcell density. We hypothesize that cell-cell interactions, presumablyhigher in the case of hybrid foam/microfibrous fabric scaffolds ascompared to the other scaffold, may have contributed to preferentialchondrocyte proliferation and differentiated functions on hybridfoam/microfibrous fabric scaffolds. The cell-cell interactions arepostulated to play a critical role in the maintenance of chondrocytephenotype. Chondrocytes are known to dedifferentiate when expanded incell monolayer. When cultured in monolayer at high cell densitychondrocytes maintain their phenotype as indicated by the expression oftype II collagen and aggrecan.

[0238] By 6 weeks, 40/60 PCL/PLA foam scaffolds, showed cartilage-likemorphology/properties localized mainly within the capsule around theconstruct. In contrast, in hybrid foam/microfibrous fabric scaffolds,thecapsule was very thin and the cell morphology within the constructs wascartilage-like and a more uniform distribution of cells was observed.

We claim:
 1. A biocompatible composite comprising a first biocompatiblefilamentous layer attached to a second biocompatible foam layer whereinthe biocompatible foam is selected from the group consisting of gradientfoams and channeled foams; wherein the gradient foam has a firstlocation and a second location wherein the biocompatible gradient foamhas a substantially continuous transition in at least one characteristicselected from the group consisting of composition, stiffness,flexibility, bioabsorption rate and pore architecture from the firstlocation to the second location of said biocompatible gradient foam andthe channeled foam has a first surface and a second surface withchannels therein.
 2. The biocompatible composite of claim 1 wherein thebiocompatible foam is bioabsorbable.
 3. The biocompatible composite ofclaim 1 wherein the biocompatible filamentous layer is bioabsorbable. 4.The biocompatible composite of claim 1 wherein the biocompatiblecomposite is made from a bioabsorbable polymer selected from the groupconsisting of aliphatic polyesters, poly(amino acids),copoly(ether-esters), polyalkylenes cxalates, polyamides,poly(iminocarbcnates), polyorthoesters, polyoxaesters, polyamidoesters,polyoxaesters containing amine groups poly(anhydrides),polyphosphazenes, biopolymers and blends thereof.
 5. The biocompatiblecomposite of claim 4 wherein the bioabsorable polymer is an aliphaticpolyester.
 6. The biocompatible composite of claim 5 wherein thealiphatic polyester is selected from the group consisting ofhcmopolymers and copolymers of lactide, lactic acid, glycolide, glycolicacid), ε-caprolactone, p-dioxanone (1,4-dioxan-2-one), trimethylenecarbonate (1,3-dioxan-2-one), alkyl derivatives of trimethylenecarbonate, δ-valerolactone, β-butyrolactone, γ-butyrolactone,ε-decalactone, hydroxybutyrate, hydroxyvalerate, 1,4-dioxepan-2-one,1,5,8,12-tetraoxacyclotetradecane-7,14-dione), 1,5-dioxepan-2-one,6,6-dimethyl-1,4-dioxan-2-one and polymer blends thereof.
 7. Thebiocompatible composite of claim 5 wherein the aliphatic polyester is anelastomer.
 8. The biocompatible composite of claim 7 wherein theelastomer is selected from the group consisting of copolymers ofε-caprolactone and glycolide; copolymers of ε-caprolactcne and(L)lactide, copolymers of p-dioxanone (1,4-dioxan-2-one) and (L)lactide,copolymers of ε-caprolactone and p-dioxanone, copolymers of p-dioxanoneand trimethylene carbonate, copolymers of trimethylene carbonate andglycolide, copolymer of trimethylene carbonate and (L)lactide and blendsthereof.
 9. The biocompatible composite of claim 5 wherein additionallypresent as a constituent of the biocompatible foam is a second aliphaticpolyester.
 10. The biocompatible composite of claim 5 whereinadditionally present as a constituent of the biocompatible filamentouslayer is a second aliphatic polyester.
 11. The biocompatible compositeof claim 4 wherein the biocompatible gradient foam has a substantiallycontinuous transition in composition from the first location to thesecond location.
 12. The biocompatible composite of claim 11 wherein thebiocompatible gradient foam has a substantially continuous transition incomposition from a first ratio of at least two different aliphaticpolyesters to a second ratio of said at least two different aliphaticpolyesters from the first surface to the second surface.
 13. Thebiocompatible composite of claim 4 wherein the biocompatible gradientfoam has a substantially continuous transition in stiffness from thefirst location to the second location.
 14. The biocompatible compositeof claim 4 wherein the biocompatible gradient foam has a substantiallycontinuous transition in bioabsorption rate from the first location tothe second location.
 15. The biocompatible composite of claim 4 whereinthe biocompatible gradient foam has a substantially continuoustransition in flexibility from the first location to the secondlocation.
 16. The biocompatible composite of claim 4 wherein thebiocompatible gradient foam has a substantially continuous transition inarchitecture from the first location to the second location.
 17. Thebiocompatible composite of claim 16 wherein the biocompatible gradientfoam has a substantially continuous transition in architecture from asubstantially spherical pore shape to a columnar pore shape from thefirst location to the second location.
 18. The biocompatible compositeof claim 16 wherein the substantially spherical pore's size is fromabout 30 μm to about 150 μm.
 19. The biocompatible composite of claim 16wherein the columnar pore's diameter is from about 100 μm to about 400μm with a length to diameter ratio of at least
 2. 20. The biocompatiblecomposite of claim 1 wherein also present in the biocompatible compositeis a therapeutic agent.
 21. The biocompatible composite of claim 1wherein additionally present is an agent is selected from the groupconsisting of antiinfectives, hormones, analgesics, anti-inflammatoryagents, growth factors, chemotherapeutic agents, anti-rejection agentsprostaglandins, RDG peptides and combinations thereof.
 22. Thebiocompatible composite of claim 21 wherein the growth factor isselected from the group consisting of bone morphogenic proteins, bonemorphogenic-like proteins, epidermal growth factor, fibroblast growthfactors, platelet derived growth factor, insulin like growth factor,transforming growth factors, vascular endothelial growth factor andcombinations thereof.
 23. The biocompatible composite of claim 1 whereinthe biccompatible compatible foam is filled with a biocomptible materialselected from the group consisting of bioabsorbable synthetic polymers,biocompatible, bicabsorbable biopolymers, biocompatible ceramicmaterials and combinations thereof.
 24. The biocompatible composites ofclaim 1 wherein the channeled foam has channels with an average lengthof at least 200 μm.
 25. The biocompatible composites of claim 24 whereinthe channels extend substantially from said first surface to said secondsurface.
 26. The biocompatible composite of claim 1 wherein thebiocompatible foam has interconnected pores formed from a compositioncontaining in the range of from about 30 weight percent to about 99weight percent ε-caprolactone repeating units.
 27. The biocompatiblecomposite of claim 26 wherein the ε-caprolactone repeating units arecopolymerized with a comonomer selected from the group consisting oflactide, lactic acid, glycolide, glycolic acid), p-dioxanone(1,4-dioxan-2-one), trimethylene carbonate (1,3-dioxan-2-one), alkylderivatives of trimethylene carbonate, δ-valerolactone, β-butyrolactone,γ-butyrolactone, ε-decalactone, hydroxybutyrate, hydroxyvalerate,1,4-dioxepan-2-one, 1,5,8,12-tetraoxacyclotetradecane-7,14-dione),1,5-dioxepan-2-one, 6,6-dimethyl-1,4-dioxan-2-one and polymer blendsthereof.
 28. The biocompatible composite of claim 26 having a firstlocation and a second location wherein the biocompatible foam has asubstantially continuous transition in at least one characteristicselected from the group consisting of composition, stiffness,flexibility, bioabsorption rate and pore architecture from the firstlocation to the second location of said biocompatible foam.
 29. Thebiocompatible composite of claim 26 wherein the interconnecting poreshave a pore size in the range from about 10 μm to about 200 μm.
 30. Thebiocompatible composite of claim 26 wherein the biocompatible foam has aporosity of in the range of from about 20 to about 98 percent.
 31. Thebiocompatible composite of claim 26 wherein the biocompatible foam haschannels.
 32. The biocompatible composites of claim 31 wherein thechannels have an average length of at least 200 μm.
 33. Thebiocompatible composite of claim 26 wherein the substantially sphericalpore's size is from about 30 μm to about 150 μm.
 34. The biocompatiblecomposite of claim 26 wherein the columnar pore's diameter is from about30 μm to about 400 μm with a length to diameter ratio of at least
 2. 35.The biocompatible composite of claim 26 wherein also present in thebiocompatible foam is a therapeutic agent.
 36. The biocompatiblecomposite of claim 1 wherein the biocompatible foam is formed with aninsert within the biocompatible foam.
 37. The biocompatible composite ofclaim 36 wherein the insert is selected from the group consisting offilms, scrims, woven textiles, knitted textiles, braided textiles,orthopedic implants, tubes and combinations thereof.
 38. Thebiocompatible composite of claim 1 wherein the biocompatible compositeis formed into a three-dimensional shaped structure.
 39. Thebiocompatible composite of claim 38 wherein the three-dimensional shapedstructure is selected from the group consisting of tubular shapes,branched tubular shapes, spherical shapes, hemispherical shapes,three-dimensional polygonal shapes, ellipsoidal shapes, toroidal shapes,conical shapes, frusta conical shapes, pyramidal shapes, both as solidand hollow constructs and combination thereof.
 40. A method for therepair or regeneration of tissue comprising contacting cells with abiocompatible composite comprising a first biocompatible filamentouslayer attached to a second biocompatible foam layer wherein thebiocompatible foam is selected from the group consisting of gradientfoams and channeled foams; wherein the biocompatible gradient foam has afirst location and a second location wherein the biocompatible gradientfoam has a substantially continuous transition in at least onecharacteristic selected from the group consisting of composition,stiffness, flexibility, bioabsorption rate and pore architecture fromthe first location to the second location of said biocompatible gradientfoam and the channeled foam has a first surface and a second surfacewith channels therein.
 41. The method of claim 40 wherein thebiocompatible composite is bioabsorbable.
 42. The method of claim 40wherein the biocompatible composite is made from a bioabsorbable polymerselected from the group consisting of aliphatic polyesters, poly(aminoacids), copoly(ether-esters), polyalkylenes oxalates, polyamides,poly(iminocarbonates), polyorthoesters, polyoxaesters, polyamidoesters,polyoxaesters containing amine groups poly(anhydrides),polyphosphazenes, biopolymers and blends thereof.
 43. The method ofclaim 42 wherein the bioabsorable polymer is an aliphatic polyester. 44.The method foam of claim 43 wherein the aliphatic polyester is selectedfrom the group consisting of homopolymers and copolymers of lactide,lactic acid, glycolide, glycolic acid), ε-caprolactone, p-dioxanone(1,4-dioxan-2-one), trimethylene carbonate (1,3-dioxan-2-one), alkylderivatives of trimethylene carbonate, δ-valerolactone,β-butyrolactone,γ-butyrolactone, ε-decalactone, hydroxybutyrate, hydroxyvalerate,1,4-dioxepan-2-one, 1,5,8,12-tetraoxacyclotetradecane-7,14-dione),1,5-dioxepan-2-one, 6,6-dimethyl-1,4-dioxan-2-one and polymer blendsthereof.
 45. The method of claim 44 wherein the aliphatic polyester isan elastomer.
 46. The method of claim 40 wherein cells are seeded ontothe biocompatible composite.
 47. The method of claim 44 wherein cellsare seeded onto the biocompatible composite.
 48. The method of claim 40wherein the biocompatible composite is implanted in an animal andcontacted with cells.
 49. The method of claim 44 wherein thebiocompatible composite is implanted in an animal and contacted withcells.
 50. The method of claim 40 wherein the biocompatible composite isseeded with cells and the biocompatible composite and cells are placedin a cell culturing device and the cells are allowed to multiply on thebiocompatible composite.
 51. The method of claim 44 wherein thebiocompatible composite is seeded with cells and the biocompatiblecomposite and cells are placed in a cell culturing device and the cellsare allowed to multiply on the biocompatible composite.
 52. The methodof claim 40 wherein the cells are selected from the group consisting ofpluripotent cells, stem cells, precursor cells and combinations thereof.53. The method of claim 40 wherein the cells are selected from the groupconsisting of myocytes, adipocytes, fibromyoblasts, ectodermal cell,muscle cells, osteoblast, chondrocyte, endothelial cells, fibroblast,pancreatic cells, hepatocyte, bile duct cells, bone marrow cells, neuralcells, genitourinary cells and combinations thereof.
 54. The method ofclaim 40 wherein the biocompatible composite contains an agent selectedfrom the group consisting of antiinfectives, hormones, analgesics,anti-inflammatory agents, growth factors, chemotherapeutic agents,anti-rejection agents, prostaglandins, RDG peptides and combinationsthereof.
 55. A method of claim 40 wherein the biocompatible composite isformed from a composition containing in the range of from about 30weight percent to about 99 weight percent ε-caprolactone repeatingunits.
 56. The method foam of claim 55 wherein the ε-caprolactonerepeating units are polymerized with a comonomer selected from the groupconsisting of homopolymers and copolymers of lactide, lactic acid,glycolide, glycolic acid), p-dioxanone (1,4-dioxan-2-one), trimethylenecarbonate (1,3-dioxan-2-one), alkyl derivatives of trimethylenecarbonate, δ-valerolactone, β-butyrolactone, γ-butyrolactone,ε-decalactone, hydroxybutyrate, hydroxyvalerate, 1,4-dioxepan-2-one,1,5,8,12-tetraoxacyclotetradecane-7,14-dione), 1,5-dioxepan-2-one,6,6-dimethyl-1,4-dioxan-2-one and polymer blends thereof.
 57. The methodof claim 55 wherein cells are seeded onto the biocompatible composite.58. The method of claim 55 wherein the biocompatible foam is implantedin an animal and contacted with cells.
 59. The method of claim 55wherein the biocompatible composite is seeded with cells and thebiocompatible composite and cells are placed in a cell culturing deviceand the cells are allowed to multiply on the biocompatible composite.60. The method of claim 59 wherein the cells are selected from the groupconsisting of pluripotent cells, stem cells, precursor cells andcombinations thereof.
 61. The method of claim 59 wherein the cells areselected from the group consisting of myocytes, adipocytes,fibromyoblasts, ectodermal cell, muscle cells, osteoblast, chondrocyte,endothelial cells, fibroblast, pancreatic cells, hepatocyte, bile ductcells, bone marrow cells, neural cells, genitourinary cells andcombinations thereof.